Cyclotides as immunosuppressive agents

ABSTRACT

The present invention relates to a pharmaceutical composition comprising a cyclotide for use in immune suppression as well as to a method for immune suppression comprising the step of administering an effective amount of a pharmaceutical composition comprising such a cyclotide to a subject in need thereof. The present invention also relates to a pharmaceutical composition comprising a cyclotide for use in treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. Likewise, the present invention also relates to a method for treating or preventing a disorder selected from the group consisting of (i) an autoimmune disorder; (ii) a hypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation. The present invention further relates to a method of screening for and/or selecting an immunosuppressive cyclotide or a mutation which results in a mutated cyclotide having an induced or enhanced immunosuppressive activity. The present invention further relates to a method of producing an immunosuppressive cyclotide or an immunosuppressive pharmaceutical composition. The present invention further relates to a mutated cyclotide having immunosuppressive activity and a pharmaceutical composition comprising the same.

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No.15/244,244, filed Aug. 23, 2016 and which is a continuation applicationof U.S. application Ser. No. 14/366,427, filed Jun. 18, 2014 and nowU.S. Pat. No. 9,453,052, and which is a § 371 U.S. National Stage Entryof International Application No. PCT/EP2012/076739, filed 21 Dec. 2012,which claims priority to European Application No. 12196918.2 filed 13Dec. 2012, and claims priority to European Application No. 11195413.7filed 22 Dec. 2011, each of which is incorporated by reference herein inits entirety. A certified copy of European Application No. 12196918.2filed 13 Dec. 2012 and European Application No. 11195413.7 filed 22 Dec.2011, was provided in, and is available in, U.S. patent application Ser.No. 14/366,427 for which certified copy is available in PAIR.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext file entitled “258-00270103-SequenceListing_ST25.txt” having a sizeof 11 kilobytes and created on Oct. 2, 2018. The information containedin the Sequence Listing is incorporated by reference herein.

The present invention relates to a pharmaceutical composition comprisinga cyclotide for use in immune suppression as well as to a method forimmune suppression comprising the step of administering an effectiveamount of a pharmaceutical composition comprising such a cyclotide to asubject in need thereof. The present invention also relates to apharmaceutical composition comprising a cyclotide for use in treating orpreventing a disorder selected from the group consisting of (i) anautoimmune disorder; (ii) a hypersensitivity disorder; and (iii) alymphocyte-mediated inflammation. Likewise, the present invention alsorelates to a method for treating or preventing a disorder selected fromthe group consisting of (i) an autoimmune disorder; (ii) ahypersensitivity disorder; and (iii) a lymphocyte-mediated inflammation.The present invention further relates to a method of screening forand/or selecting an immunosuppressive cyclotide or a mutation whichresults in a mutated cyclotide having an induced or enhancedimmunosuppressive activity. The present invention further relates to amethod of producing an immunosuppressive cyclotide or animmunosuppressive pharmaceutical composition. The present inventionfurther relates to a mutated cyclotide having immunosuppressive activityand a pharmaceutical composition comprising the same.

Naturally-occurring circular peptides with potential pharmaceuticalapplications have been found in various organisms (summarized in Craik,2006, Science, 311, 1563-1564), such as bacteria (e.g., bacteriocinAS-48 (Martinez-Bueno, 1994, J Bacteriol, 176, 6334-6339) and MicrocinJ25 (Rosengren, 2003, J Am Chem Soc, 125, 12464-12474)), plants (e.g.sunflower trypsin inhibitors (Luckett, 1999, J Mol Biol, 290, 525-533;Mylne, 2011, Nat Chem Biol, 7, 257-259)) and animals (e.g. rhesus monkeyθ-defensins (Tang, 1999, Science, 286, 498-502)). One of the largest,but mostly unexplored, group of natural circular peptides are the plantcyclotides (Gruber, 2010, Curr Pharm Des, 16, 3071-3088).

In general, cyclotides are head-to-tail cyclized peptides representingan abundant and diverse group of (ribosomally-) synthesized plantpeptides containing a cyclic cystine-knotted structure. Moreover,cyclotides are a natural combinatorial library of circular cystine-knotpeptides with great stability. Cyclotides are explored for theirdistribution in plants, although little is known about the individualpeptide content of a single species.

The circular cyclotide chain usually consists of ˜30 amino acids,including six conserved cysteines that form three disulfide bondsarranged in a cyclic cystine-knot (CCK) motif (Craik, 1999, J Mol Biol,294, 1327-1336), whereas the inter-cysteine sequences can tolerate awide range of residue substitutions and, hence, the cyclotide scaffoldmay serve as a natural combinatorial peptide template (Clark, 2006,Biochem J, 394, 85-93).

The remarkable structural features make cyclotides extremely resistantto enzymatic, chemical and thermal degradation (Colgrave, 2004,Biochemistry, 43, 5965-5975). In contrast to non-ribosomal synthesizedplant metabolites, cyclotides are true gene products and theirbiosynthesis involves ribosomal precursor synthesis, enzymaticprocessing (Gillon, 2008, Plant J, 53, 505-515; Saska, 2007, J BiolChem, 282, 29721-29728) and protein folding events (Gruber, 2007, J BiolChem, 282, 20435-20446). Furthermore, cyclotides possess a wide range ofbiological activities, e.g., insecticidal (Barbeta, 2008, Proc Natl AcadSci USA, 105, 1221-1225; Gruber, 2007, Toxicon, 49, 561-575),nematocidal (Colgrave, 2008, Biochemistry, 47, 5581-5589; Colgrave,2009, Acta Trop, 109, 163-166), anti-fouling (Goransson, 2004, J NatProd, 67, 1287-1290), and anti-HIV (Wang, 2008, J Nat Prod, 71, 47-52;Ireland, 2008, Biopolymers, 90, 51-60) activities, as well ascytotoxicity to lymphoma cell lines (Svangard, 2004, J Nat Prod, 67,144-147; Lindholm, 2002, Mol Cancer Ther, 1, 365-369).

The discovery of the first cyclotide, kalata B1, was based on itspresence in tea/extract from the Rubiaceae species Oldenlandia affinis(R&S) DC. used in African indigenous medicine to accelerate childbirth(Gran, 1970, Medd Nor Farm Selsk, 12, 173-180; 1973, Acta PharmacolToxicol (Copenh), 33, 400-408; Gruber, 2011, Planta Med, 77, 207-220).The plant O. affinis (Rubiaceae) is commonly known to scientists in thefield of ethnopharmacology and peptide chemistry as a prototypicalsource of cyclotides.

Since their discovery in the coffee-family (Rubiaceae), cyclotides havebeen extensively studied in the violets (Violaceae), and have recentlybeen found in legumes (Fabaceae) (Poth, 2011, Proc Natl Acad Sci USA,108, 10127-10132; Poth, 2011, ACS Chem Biol, 6, 345-355; Nguyen, 2011, JBiol Chem, 286, 24275-24287). There is an increasing effort to screenplants of different families for the occurrence and distribution ofcyclotides. Today it is evident that many other cyclotides exist.Recently it has been estimated that there are at least 50,000 novelcyclotides to be discovered in Rubiaceae (Gruber, 2008, Plant Cell, 20,2471-2483) and another ˜9,000 in Violaceae (Simonsen, 2005, Plant Cell,17, 3176-3189; Trabi, 2004, J Nat Prod, 67, 806-810), but researchersare only at the beginning to understand their variety and distributionin plants (Gruber, 2010, Biopolymers, 94, 565-572). Biologically,cyclotides are mainly explored for applications in agriculture and drugdesign due to their enormous stability (Craik, 2001, Toxicon, 39,1809-1813; Craik, 2007, Curr Opin Drug Discov Devel, 10, 176-184; Craik,2006, Biopolymers, 84, 250-266; Craik, 2001, Toxicon, 39, 43-60). Thecyclotide kalata B1 has earlier been reported to cause hemolysis andmembrane disruption at concentrations above ˜50 μM (Barry, 2003,Biochemistry, 42, 6688-6695; Henriques, 2011, J Biol Chem, 286,24231-24241).

With respect to the therapeutic applications of cyclotides, thescientific and patent literature is primarily related to the use ofcystine knot scaffolds for the production of peptide-based drugs. Forexample, U.S. Pat. No. 7,960,340 B2 is based on the concept that thecyclotide molecular framework is ultra-stable and that it is possible tomodify loops of the framework by replacing them with pharmaceuticallyrelevant bioactive sequences, thereby stabilizing this bioactivesequences. Several recent papers have reported examples of thiscyclotide grafting strategy. Subsequently, various studies appliedcyclotides as scaffolds for therapeutically active peptides (see, forinstance, Smith, 2011, Expert Opin. Ther. Patents 21, 1657-1672;Gunasekera, 2008, J Med Chem, 51, 7697-704; Thonbyoo, 2008, Org BiomolChem, 6, 1462-70; and Cemazar (20th American-Peptide-Society Symposium;Montreal, CANADA, Jun. 26-30, 2007, Biopolymers 88, 4, SI, 2007, 523.Examples include the development of an inhibitor of angiogenesis withapplications in cancer therapy by the grafting of an antiangiogenicsequence onto the cyclotide kalata B1 (Gunasekera; 2008; J Med Chem; 51;7697-704) and the development of an inhibitor of a protease fromfoot-and-mouth disease virus onto the MCoTI-II cyclotide framework(Thonbyoo; 2008; Org Biomol Chem; 6; 1462-70). It has also been shownthat cyclotides have the activity of inhibiting tryptase, the majorsecretory protease of human mast cells (WO 06032436). Furthermore,cyclotides were shown to exhibit cytotoxic activities and theirselective toxicity to cancer cell lines has opened the possibility ofanti-cancer applications (Hermann; 2008; Phytochemistry; 69; 939-52,Lindholm; 2002; Mol Cancer Ther; 1; 365-9, Svangard; 2004; J Nat Prod;67; 144-7; Burman; 2010; Biopolymers Pept Sci; 94; 626-34; Gerlach;2010; Biopolymers; 94; 617-25). However, cytotoxicity effects are knownto cause side effects.

The body's immune system is a very powerful weapon against pathogens,but malfunctioning can cause an over-reactivity of this defensemachinery and, in some instances, lead to auto-immune diseases, such asrheumatoid arthritis (RA) or Crohn's disease. Immunosuppression, thetargeted reduction of the activation or efficacy of the immune system,is a potential approach for the treatment of these conditions. BecauseT-lymphocytes have the greatest powerful impact during defence responseof the immune system, most immunosuppressive medications aim to act onthese cells. One of the clinically used immunosuppressive drugs ofchoice to treat or suppress an “over-activity” of lymphocytes, forexample after transplantation surgery or in cases of severe RA, is thefungal, cyclic non-ribosomal peptide cyclosporine A (de Mattos, 2000, AmJ Kidney Dis, 35, 333-346; Matsuda, 2000, Immunopharmacology, 47,119-125; Schreiber, 1991, Science, 251, 283-287). However, cyclosporineA has many and sometimes severe side effects (de Mattos, 2000, Am JKidney Dis, 35, 333-346). In addition, leaf extracts of Betula pendulahave been traditionally used for treating RA or osteoarthritis.

Beside this, further problems have to be faced when trying to suppressthe immune system. For example, the proof of anti-proliferative effectsby holding the cells in an “inactive” state at which they are stillviable, but aren't able to grow and proliferate, without causing celldeath is a crucial precondition to classify a substance asimmunosuppressant, because cytotoxicity would cause side effects.Moreover, therapeutic peptides often lack oral bioavailability due tofast degradation upon ingestion and have poor drug permeation due totheir hydrophilic nature.

Thus, the technical problem underlying the present invention is theprovision of improved means and methods for suppressing the immunesystem/for immunosuppression and for treating/preventing correspondingdiseases/disorders.

The technical problem is solved by providing the embodimentscharacterized herein and in the appended claims.

In the context of the present invention, the proof of principle was madethat cyclotides and related cystine-knot peptides or cyclotidemutants/variants can be used for suppressing the immune system/immunesuppression, as immunosuppressive agents or for thesuppression/reduction of the efficacy of the immune system. Thisprovides for the possibility to make use of the superior, advantageous(pharmacological) features of cyclotides also in the field ofsuppression of the immune system/immune suppression.

In particular, it was surprisingly found in the context of thisinvention that there are cyclotides existing which are capable todecrease or arrest proliferation of (activated) immune cells/cells ofthe immune system (for example peripheral blood mononuclear cells (PBMC)or (T-)lymphocytes). Furthermore, the anti-proliferative effect inducedby the cyclotides was shown not to cause cell death by either apoptosisor necrosis, but to inhibit the growth of the immune cells in acytostatic fashion. In addition, vaccination with a cyclotide (forexample kalata B1) resulted in a reduction in the incidence and severityof Experimental Autoimmune Encephalomyelitis (EAE) in an EAE mouse modelfor multiple sclerosis (MS). Moreover, it was shown that vaccinationwith a cyclotide (for example kalata B1), leads to the production of ananti-inflammatory T-cell response.

Accordingly, the present invention relates to a pharmaceuticalcomposition comprising (as the active ingredient) a cyclotide, inparticular an anti-immune cell-proliferative cyclotide, for use inimmune suppression. Moreover, the present invention relates to a methodfor immune suppression comprising the step of administering an effectiveamount of a pharmaceutical composition comprising (as the activeingredient) a cyclotide, in particular an anti-immune cell-proliferativecyclotide, to a subject/patient in need thereof.

In the context of the present invention the immunosuppressive propertiesof (plant) cyclotides were characterized for the first time. Combinedwith the unique structural features and enormous stability ofcyclotides, these results open new avenues for the application of nativeand synthetically-optimized (plant) cyclotides in immune-relateddisorders, in particular in immune suppression.

In particular, it was demonstrated in the context of the presentinvention that there are anti-proliferative effects of an extract fromthe coffee-family plant O. affinis to cells of the human immune system(for example lymphocytes). In addition, kalata B1 was specificallyidentified as one active component responsible for the observedcytostatic effects. Moreover, it was demonstrated that, in a definedconcentration range, no cell death by apoptosis or necrosis was caused.Moreover, it was shown that EAE mice treated with a cyclotide (forexample kalata B1) displayed significantly milder clinical signs anddisplayed improvement in disease severity (see, for example, FIGS. 10Aand B). In addition, vaccination with a cyclotide (for example kalataB1) leads to the production of an anti-inflammatory T-cell response.

The presented results have further impact to the general field ofcystine-knot peptides and greatly enhance the possibilities for theirpotential therapeutic applications. The oral bioavailability ofcyclotides and cystine-knot peptides, the availability of recombinantand synthetic production techniques as well as the plasticity of thecystine-knot framework, which is amenable to a wide range of amino acidsubstitutions, provides a promising basis for future mechanistic studiesof their activity on immune cells (e.g. lymphocytes) and in vivoapplications (for example in model systems related to malfunctioning ofimmune cells in general and in particular, the over-reactivity oflymphocytes).

As documented herein below and in the appended examples, theanti-proliferative effects of cyclotides, in particular plantcyclotides, on primary cells of the human immune system (primary humanPBMC or T-lymphocytes) was shown using biological and immunologicalendpoints in cell-based test systems. It was further shown that theeffects have a defined concentration range and were not due to cytotoxiceffects. More particular, LC-MS quantification of the active O. affinisplant extract triggered the characterization of the anti-proliferativeactivity of kalata B1, one of the most abundant cyclotides in thisextract, on primary activated human lymphocytes. For this purpose, acrude O. affinis cyclotide extract was analyzed using an alternativepeptidomics workflow and a rapid technique for the characterization ofcyclotides in plant.

It was shown in the appended examples that a cyclotide (for example thekalata B1-mutant cyclotide T20K, a cyclotide comprising SEQ ID NO. 7; 4μM) is capable of reducing the expression level of the IL-2 receptor andthe IL-2 production. The magnitude of the effect was similar to thetreatment with cyclosporine A (5 μg/ml) and this anti-proliferativeeffect of the cyclotide could be reversed by addition of exogenous IL-2.Furthermore, it was shown that the cyclotide reduced the release ofeffector molecules IFN-gamma and TNF-alpha in PBMCs, however thisreduction was only of transient nature. This is in contrast toCsA-treatment, which led to a retained reduction of TNF-alpha andIFN-gamma over time. A reduction in IL-2 release upon treatment withcyclotides (for example with the kalata B1-mutant cyclotide T20K, acyclotide comprising SEQ ID NO. 7; 200 μg/100 μl/mouse) was also shownin vivo (for example in an EAE mouse model (C57BL/6J)).

Without being bound by theory, the cyclotide-mediated anti-proliferativeeffect is mediated through an IL-2-depending mechanism. The effectorfunctions of (activated) PBMC (for example lymphocytes) were alsoreduced by cyclotide treatment (for example by treatment with T20K).

Moreover, the effect on IL-2 synthesis and IL-2 receptor expression maybe directly influenced by cyclotides or independently mediated. Theherein defined cyclotides may have a similar mode of action as comparedto CsA. CsA is known to directly influence the IL-2 production. Further,CsA is able to form a complex with cyclophilin and the CsA-cyclophilincomplex can bind to calcineurin and inhibit its function inCa-signalling. This leads to a reduced NFATc transcription and henceIL-2 synthesis. The immunosuppressive action of CsA hence requires CsAto enter the cells and form a direct contact with cyclophillin andcalcineurin. As shown in the appended Examples T20K can enter T-cells,i.e. it can pass (actively or passively) the membrane (see FIG. 24).Accordingly and without being bound by theory, T20K may interact with anextracellular target or transporter or may enter the T-cell passivelyand interact with an intracellular target. Also a combination ofextracellular and intracellular activity of T20K is possible. Withoutbeing bound by theory, the herein defined cyclotides, in particularkalata B1 or T20K, may be able to enter cells and affect the IL-2synthesis in CsA manner or may remain on the outside of its target cellsand lead to a change in the membrane potential by interaction withsurface molecules, receptors or ion-channels. Most importantly, theherein defined cyclotides, in particular kalata B1 or T20K, may interactwith a T-cell receptor.

Without being bound by theory, the anti-proliferative mechanism may bedue to direct interaction of the herein defined cyclotides with the IL-2receptor (for example, T20K is able to down-regulate the IL-2alpha-chain CD25 receptor expression on the surface of PBMCs as shown inthe appended examples). For example, and also without being bound bytheory, binding of the herein defined immunosuppressive cyclotides tothe IL-2 alpha-chain may occupy the interaction site for binding of thebeta- and gamma-chain and hence inhibit complex formation. However, thisIL-receptor complex formation is important for activation of theT-lymphocyte in order to receive signals from released IL-2. Only afterbinding of IL-2 to its receptor, the T-lymphocytes will initiate anormal proliferation. If binding is inhibited, for example by abovedescribed mechanism, the T-cells remain in a non-proliferative state.One drug on the pharmaceutical market, i.e. Simulect®, is used asanti-proliferative agent on the basis of CD25 receptor interaction. Theactive principle is a chimeric monoclonal antibody, Basiliximab, whichbinds to the IL-2 receptor alpha chain and hence inhibits binding ofendogenous IL-2.

In the context of the present invention, the anti-proliferative andcytotoxic effects of a crude O. affinis cyclotide-containing plantextract towards activated primary human lymphocytes was characterized.To identify the individual molecular peptide components of thisimmunosuppressive cyclotide mixture, biological in vitro analysis werecombined with chemical characterization of the content of individualpeptides (cyclotides) in the crude extract of this plant using anoptimized rapid peptidomics workflow.

In particular, an optimized protocol for the analysis ofcyclotide-containing plant extracts by combining nanoflow LC-MS/MS andautomated database analysis was used to determine the content ofdistinct peptides (by molecular weight and peptide sequence) in thecyclotide-containing plant O. affinis.

The combination of nano LC-MS/MS and LC-MS reconstruction, as well asautomated database searching (e.g. using the ERA tool (Colgrave, 2010,Biopolymers. 94, 592-601)) is a rapid and useful technique for theidentification of cyclotides in crude extracts.

Compared to an earlier study from Plan (2007, ChemBioChem, 8,1001-1011), which described the first cyclotide fingerprint of O.affinis using classical peptide purification via analytical HPLC andoffline MS/MS sequencing, 8 additional known cyclotides were identifiedand shown to be able to provide a list of ˜50 peptide massescorresponding to cyclotides of which some can be identified by peptidefingerprint analysis in CyBase (the cyclotide database (Wang, 2008,Nucleic Acids Res, 36, D206-210)). This suggests that the number ofcyclotides to be found in a single species may be >70 and is, therefore,at least twice the number than earlier anticipated (on average 34cyclotides per species (Gruber, 2008, Plant Cell, 20, 2471-2483). This,of course, has a huge impact on the determination of the overall numberof cyclotides in the plant kingdom and consequently would lead to anecessary revision of the number of novel cyclotides to be discovered inplants.

Using the above described improved peptidomics workflow, nearly allcurrently known cyclotides and an even greater number of novel peptidemasses corresponding to other known or novel cyclotides (by molecularweight) could be identified in crude cyclotide extract from the plant O.affinis. The cyclotides kalata B1 and kalata B2 were found to be themain peptide components, accounting for approx. 34% of the overallcyclotide content in O. affinis.

By using flow cytometric-based forward-side-scatter analysis, it wasfurther demonstrated that the cyclotide-containing extract exhibits adose-dependent (50-100 μg/mL) decrease of activated proliferating PBMCcompared to untreated stimulated control (FIGS. 2A and B).Simultaneously, a constant content of viable, resting PBMC, withoutaccumulation of dead cells was observed, showing that the appliedconcentrations of the cyclotide extract are not harmful to the cells.

Several additional characteristics regarding drug delivery conduce tothe above described immunosuppressant potential of cyclotides: (i)retained activity upon oral administration as tea/extract (in humans)(Gran, 1970, Medd Nor Farm Selsk, 12, 173-180), (ii) great stability inplasma and against gastro-intestinal proteases (Colgrave, 2004,Biochemistry, 43, 5965-5975; Colgrave, 2005, J Chromatogr A, 1091,187-193) and (iii) the presence of surface-exposed hydrophobic patches(Clark, 2006, Biochem J, 394, 85-93).

Generally, therapeutic peptides often lack oral bioavailability due tofast degradation upon ingestion and have poor drug permeation due totheir hydrophilic nature (Vlieghe, 2010, Drug Discov Today, 15, 40-56;Werle, 2007, Int J Pharm, 332, 72-79). Cyclotides and relatedcystine-knot peptides are likely to overcome these problems (Kolmar,2009, Curr Opin Pharmacol, 9, 608-614). As corresponding proofs ofconcept there are two examples in the literature: (i) asynthetically-engineered cyclic conotoxin has recently been confirmed asan oral active circular peptide drug for the treatment of neuropathicpain in vivo (Clark, 2010, Angew Chem Int Ed Engl, 49, 6545-6548), and(ii) a synthetic cyclotide containing the sequence motif of a bradykininB1 antagonist has been engineered based on the native kalata B1 peptidetemplate and has been confirmed to be orally active and bioavailable ina mouse model of inflammatory pain (Wong, 2012, Angew Chem Int Ed Engl,51(23), 5620-4). Another feature of cyclotides with respect toapplications as peptide drugs is that they are synthesized gene products(Jennings, 2001, Proc Natl Acad Sci USA, 98, 10614-10619) and cantherefore be produced in large quantity by recombinant techniques(Kimura, 2006, Angew Chem Int Ed Engl, 45, 973-976) or in plantsuspension cultures (Seydel, 2007, Appl. Microb. Biotechnol., 77,275-284). These cyclotide production techniques and the availability ofsolid-phase peptide synthesis strategies (Clark, 2010, Biopolymers, 94,414-422) offer opportunities for the optimization of thecyclotide-framework, which is amenable to a wide range of amino acidsubstitutions (see also FIG. 1).

Not at least, the proof of anti-proliferative effects by holding thecells in an “inactive” state at which they are still viable, but aren'table to proliferate, without causing cell death, in a certain dose rangeis a crucial precondition to classify a substance as immunosuppressant,because cytotoxicity would cause side effects.

It was further demonstrated in the context of the present inventionthat, upon treatment (for example of an EAE mouse model (C57BL/6J)) withcyclotides (for example with the kalata B1-mutant cyclotide T20K, acyclotide comprising SEQ ID NO. 7; 200 μg/100 μl/mouse), the clinicalscore (for example the EAE score) significantly decreases (for examplethe weight of EAE mice). Importantly, cyclotide treatment does not causea cytotoxic effect since cyclotide treatment does not lead to a bodyweight reduction.

In general, the meaning of the term “cyclotide” is known in the art andthe term “cyclotide” is correspondingly used herein. In particular,“cyclotides” as used herein are head-to-tail cyclized peptides whichcyclotide chain includes six conserved cysteine residues capable to formthree disulfide bonds arranged in a cyclic cystine-knot (CCK) motive.The inter-cysteine sequences of a cyclotide can tolerate a wide range ofresidue substitutions (see, for example, Clark, loc. cit. and FIG. 1).In one aspect, the term “cyclotide” used herein refers to cyclotides asdescribed in Craik (1999, loc. cit.), Clark (2006, loc. cit.) and, inparticular, in U.S. Pat. No. 7,592,533 B1.

In particular, a cyclotide to be used in the context of this inventioncomprises an amino acid sequence capable of forming a cyclic backbonewherein said cyclic backbone comprises the structure of formula I:Cyclo(C[X₁ . . . X_(a)]C[X^(I) ₁ . . . X^(I) _(b)]C[X^(II) ₁ . . .X^(II) _(c)]C[X^(III) ₁ . . . X^(III) _(d)]C[X^(IV) ₁ . . . X^(IV)_(e)]C[X^(V) ₁ . . . X^(V) _(f)])  (I)

wherein

-   -   (i) C is cysteine;    -   (ii) each of [X₁ . . . X_(a)], [X^(I) ₁ . . . X^(I) _(b)],        [X^(II) ₁ . . . X^(II) _(c)], [X^(III) ₁ . . . X^(III) _(d)],        [X^(IV) ₁ . . . X^(IV) _(e)], and [X^(V) ₁ . . . X^(V) _(f)]        represents one or more amino acid residues, wherein each one or        more amino acid residues within or between the sequence residues        may be the same or different; and    -   (iii) a, b, c, d, e, and f represent the number of amino acid        residues in each respective sequence and each of a to f may be        the same or different and range from 1 to about 20.

Preferably, a is 3 to 6, b is 4 to 8, c is 3 to 10, d is 1, e is 4 to 8,and/or f is 5 to 13.

Preferably, the cyclotide to be used herein comprises the amino acidstretch of formula II (SEQ ID NO. 17)Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Xxx₁-Cys-Xxx₁-Trp-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁  (II),wherein Xxx₁, Xxx₂ and Xxx₃ is any amino acid, non-natural amino acid orpeptidomimetic, preferably an aliphatic amino acid. In particular, Xxx₂may be any amino acid, non-natural amino acid or peptidomimetic but notLys and/or Xxx₃ may be any amino acid, non-natural amino acid orpeptidomimetic but not Ala or Lys. Preferably, Xxx₂ and/or Xxx₃ offormula II are not mutated at all. More particular, Xxx₁ may be Gly,Thr, Ser, Val, Ile, Asn, Asp or, preferably, Lys, Xxx₂ may be Thr,and/or Xxx₃ may be Val or Phe. Even more particular, Xxx₁ at position 1of formula II may be Gly, Xxx₁ at position 18 of formula II may be Lysor, preferably, Gly, Xxx₁ at position 20 of formula II may be Thr, Seror, preferably, Lys, Xxx₁ at position 22 of formula II may be Ser orThr, Xxx₁ at position 25 of formula II may be Val or Ile, Xxx₁ atposition 29 of formula II may be Asn, Asp or, preferably, Lys, Xxx₂ offormula II may be Thr and/or Xxx₃ of formula II may be Val or Phe.

The specifically defined amino acid residues of formula II may also varydepending on the particular (type of) cyclotide. Hence, what has beensaid with respect to Xxx₁, Xxx₂ and/or Xxx₃, does not only apply toformula II but also to the corresponding amino acid residues of othercyclotides not comprising the particular amino acid stretch of formulaII. In this context, “corresponding” particularly means amino acidresidues at the same or similar position(s).

Non-limiting specific examples of a cyclotide to be used according tothis invention is a cyclotide comprising:

-   (i) an amino acid sequence selected from the group consisting of SEQ    ID NOs: 7, 5, 1, 4, 6, 2 and 3;-   (ii) an amino acid sequence encoded by a nucleotide sequence    selected from the group consisting of SEQ ID NOs: 11, 15, 12 and 16;-   (iii) an amino acid sequence encoded by a nucleotide sequence    encoding an amino acid sequence selected from the group consisting    of SEQ ID NOs: 7, 5, 1, 4, 6, 2 and 3; or-   (iv) an amino acid sequence that is at least 70%, preferably at    least 80%, more preferably at least 90%, even more preferably at    least 95%, even more preferably at least 98% and even more    preferably at least 99% identical to any amino acid sequence of (i)    to (iii).

Further, non-limiting examples of cyclotides to be used are cyclotidesconsisting of a head-to-tail cyclized form of an amino acid sequence asdefined in any of (i) to (iv), supra.

In a preferred embodiment, the cyclotide to be used is kalata B or akalata B-type cyclotide. In an even more preferred embodiment, thecyclotide is kalata B2 or, most preferably, kalata B1. The cyclotideskalata B1 and B2 differ by only five amino acid positions (see FIG. 6),namely Val to Phe (loop 2) and conservative replacements of Thr to Ser(loop 4), Ser to Thr (loop 5), Val to Ile (in loop 5) and Asn to Asp (inloop 6) in kalata B2. These substitutions have no significant structuralconsequences (RMSD_(backbone kB1/kB2)=0.599 Å, see FIG. 6) and the twopeptides have a similar bioactivity profile (Gruber, 2007, Toxicon, 49,561-575).

It will be understood that for the various cyclotides to be used in thecontext of the present invention a certain flexibility and variabilityin the primary sequence, i. e. the amino acid sequence backbone, ispossible, as long as the overall secondary and tertiary structure of therespective peptides, which is defined by at least some fixed amino acidresidues and by their spatial arrangement, is ensured (see, e.g.,formulas I and II, supra).

Based on the teaching provided herein, the skilled person is, one theone hand, readily in the position to find out/identify correspondingmutants/variants of the cyclotides which act according to the invention.One the other hand, the skilled person is able to test whether a givencyclotide mutant/variant still has the desired function, for example atleast one of the functions as described herein elsewhere. Correspondingexperimental guidance for such tests, i.e. respective assays, areexemplarily provided and described herein, particularly in the appendedexamples.

Hence, in one aspect, the present invention also relates to the use ofmutant or variant forms of the herein defined (native) cyclotides, inparticular to the use of mutant or variant forms of the cyclotides asdepicted in Table 1, more particular of mutant or variant forms ofkalata B2 or, preferably, kalata B1. The mutant or variant forms may be(synthetically) optimized, i.e. they may be better suited forimmunosuppression as compared to their non-mutant/non-variant form.Non-limiting examples of mutant/variant forms of cyclotides are thecyclotides as depicted in Table 1, wherein the same mutations as in anyone of SEQ ID NO: 3 to 7 have been performed or corresponding mutationsat amino acid positions which correspond to the amino acid positionswhich have been mutated in any one of SEQ ID NO: 3 to 7 have beenperformed.

If not mentioned differently, the term “cyclotide(s)” when used hereinis envisaged to also encompass “cyclotide mutant(s)/variant(s)”.Non-limiting examples of mutant/variant/modified cyclotides according tothis invention are given in section (iv), supra or are cyclotidesconsisting of a head-to-tail cyclized form of an amino acid sequence asdefined in section (iv), supra. Further examples of mutant/variantcyclotides are cyclotides comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 3 to 7 or cyclotides consistingof a head-to-tail cyclized form of an amino acid sequence selected fromthe group consisting of SEQ Id NOs: 3 to 7.

As to the mutants/variants of the cyclotides it is, for example,envisaged that one or more amino acids of said peptides are replaced byother one or more naturally-occurring or synthetic amino acids. In thiscontext, it is preferred that this/these amino acid exchange(s) is/are(a)conservative amino acid exchange(s), i.e. that the replacement aminoacid belongs to the same category of amino acids than the amino acid tobe replaced. For example, an acidic amino acid may be replaced byanother acidic amino acid, a basic amino acid may be replaced by anotherbasic amino acid, an aliphatic amino acid may be replaced by anotheraliphatic amino acid, and/or a polar amino acid may be replaced byanother polar amino acid.

It is particularly envisaged that the amino acid exchanges which lead tomutants/variants of the disclosed cyclotides are such that the patternof polarity and charge within the tertiary structure of the resultingmutant/variant still (substantially) mimics/corresponds to thethree-dimensional structure of the respective cyclotide.

Further examples of mutant or variant cyclotides are kalata B1 or kalataB2 (or the disclosed mutants/variants thereof) or a cyclotide consistingof a head-to-tail cyclized form of the amino acid sequence of SEQ ID NO:1 or 2 having

-   (i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of its acidic amino    acid residues replaced by a different amino acid residue selected    from the group consisting of acidic amino acid residue;-   (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of its basic amino    acid residues replaced by a different amino acid residue selected    from the group consisting of basic amino acid residues; and/or-   (iii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of its aliphatic    amino acid residues replaced by a different amino acid residue    selected from the group consisting of aliphatic amino acid residues.

Other mutant/variant cyclotides comprise the amino acid stretch offormula II, but having

-   (i) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the (remaining    specific) acidic amino acid residues replaced by a different amino    acid residue selected from the group consisting of acidic amino acid    residues;-   (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the (remaining    specific) basic amino acid residues replaced by a different amino    acid residue selected from the group consisting of basic amino acid    residues; and/or-   (iii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the (remaining    specific) aliphatic amino acid residues replaced a different amino    acid residue selected from the group consisting of aliphatic amino    acid residues.

In general, the meaning of the term “amino acid” or “amino acid residue”is known in the art and is used herein accordingly. Thereby, it is ofnote that when an “amino acid” is a component of a peptide/protein theterm “amino acid” is used herein in the same sense than “amino acidresidue”.

Particularly, an “amino acid” or “amino acid residue” as referred toherein is envisaged to be a naturally-occurring amino acid, morepreferably a naturally-occurring L-amino acid. However, albeit lesspreferred, an “amino acid” or “amino acid residue” in context of thisinvention may also be a D-amino acid or a non-naturally-occurring (i.e.a synthetic) amino acid, like, for example, norleucine, β-alanine, orselenocysteine.

Also known in the art is the meaning of the terms “acidic aminoacid(s)”, “basic amino acid(s)”, “aliphatic amino acid(s)” and “polaramino acid(s)” (see, for example, Stryer, Biochemie, Spectrum Akad.Verlag, 1991, Item I. 2.). These terms are correspondingly usedthroughout this invention. Thereby, the particular provisos given hereinwith respect to the cyclotides of the invention also apply.

Particularly, the term “acidic amino acid(s)” as used herein is intendedto mean an amino acid selected from the group comprising Asp, Asn, Glu,and Gln, the term “basic amino acid(s)” as used herein is intended tomean an amino acid selected from the group comprising Arg, Lys and His,the term “aliphatic amino acid(s)” as used herein is intended to meanany amino acid selected from the group comprising Gly, Ala, Ser, Thr,Val, Leu, Ile, Asp, Asn, Glu, Gln, Arg, Lys, Cys and Met, and the term“polar amino acid(s)” as used herein is intended to mean any amino acidselected from the group comprising Cys, Met, Ser, Tyr, Gln, Asn and Trp.

In a preferred embodiment, the cyclotides and mutant/variant cyclotidesto be used in accordance with the present invention are cyclotideshaving at least one of their amino acid residues corresponding to Xxx₁of formula II, preferably corresponding to Xxx₁ at position 20 and/or 29of formula II, replaced by (a) different amino acid residue(s).Likewise, the cyclotides and mutant/variant cyclotides to be used inaccordance with the present invention may also be cyclotides having atleast one of their amino acid residues corresponding to amino acidposition 1, 18, 20, 22, 25 and/or 29, preferably corresponding to aminoacid position 20 and/or 29, replaced by (a) different amino acidresidue(s). In this context, “corresponding to” particularly means thesame amino acid amino acid residue(s) and/or at the same or similarposition(s). Such (a) different amino acid residue(s) may, for example,be useful for labelling the respective mutant/variant cyclotides. Anon-limiting example of such (a) different amino acid residue(s) is Lys.Non-limiting examples of respective mutant/variant cyclotides aremutant/variant cyclotides comprising or consisting of (a head-to-tailcyclized form of) a amino acid sequence of SEQ ID NO: 4 to 7, whereinSEQ ID NOs. 5 or 7 are preferred.

In a specific aspect, the mutant/variant cyclotides to be used accordingto the invention are cyclotides not having replaced one or more of theiramino acid residues lying between the “first” and the “second” Cys(corresponding to the “first” and “second” Cys, respectively, asdepicted in formula I, supra) and/or between the “second” and the“third” Cys (corresponding to the “second” and “third” Cys,respectively, as depicted in formula I, supra).

Preferably, in such mutant/variant cyclotides none of the amino acidresidues flanking the “second” Cys, in particular neither the amino acidresidue next to the “second” Cys in the N-terminal direction of formulaI nor the amino acid residue next to the “second” Cys in the C-terminaldirection of formula I, are replaced by another amino acid residue, inparticular not by an Lys or Ala residue.

It is preferred that the used cyclotides and mutants/variants thereoflack sites susceptible for hydrolysis or cleaving proteases, like, forexample, serum proteases. The meanings of the terms “hydrolysis” and“(serum) proteases” and the structure of the sites are well known in theart.

In a preferred aspect, in the mutant/variant cyclotides to be used inaccordance with the present invention, in particular in themutant/variant cyclotides more specifically defined herein elsewhere(for example, the mutant/variant cyclotides as defined in items (iv) and(i) to (iii), supra, or items (i) to (xi), infra), none of the (six) Cysresidues is replaced by another amino acid residue.

However, with respect to the mutants/variants of the cyclotides, one ormore of the (six) Cys residues, in particular the herein defined Cys,may also be replaced by (an)other amino acid(s), as long as thereplacement still leads to an individual intramolecular linkage, likethat of a disulphide bond, within the cyclopeptide, i.e. to a correctmimicry of the native cyclotide. Such amino acid may, inter alia, be anon-naturally-occurring amino acid, like a non-naturally-occurring aminoacid having an —SH group able to form a disulphide bond. However, it ispreferred herein that the Cys, in particular the Cys given in formula I,above, is a naturally-occurring amino acid, preferably Cys itself.

It will also be acknowledged by the ones skilled in the art that one orseveral of the amino acids forming the cyclotide to be employedaccording to the present invention may be modified. In accordancetherewith any amino acid as used/defined herein may also represent itsmodified form. For example, an alanine residue as used herein maycomprise a modified alanine residue. Such modifications may, amongothers, be a methylation or acylation, or the like, whereby suchmodification or modified amino acid is preferred as long as the thusmodified amino acid and more particularly the cyclotide containing saidthus modified amino acid is still functionally active as defined herein.Respective assays for determining whether such a cyclotide, i. e. acyclotide comprising one or several modified amino acids, fulfils thisrequirement, are known to the one skilled in the art and, among others,also described herein, particularly in the example part.

The invention also provides the use of derivatives of the disclosedcyclotides such as salts with physiologic organic and anorganic acidslike HCl, H₂SO₄, H₃PO₄, malic acid, fumaric acid, citronic acid,tatratic acid, acetic acid.

It is particularly envisaged that the herein defined cyclotides, and theherein defined mutant cyclotides and variant cyclotides (see, forexample, item (iv), supra) have at least one of the desired functionsaccording to this invention, in particular, one of the functions asmentioned in items (i) to (xii) herein below. This/these function(s)make the cyclotides and cyclotide mutants/variants beingimmunosuppressive cyclotides and immunosuppressive cyclotidemutants/variants in accordance with the present invention.

In one aspect, the cyclotides and cyclotide mutants/variants to be usedin accordance with this invention and as defined herein

-   (i) are anti-proliferative cyclotides, i.e. have an (dose-dependent)    anti-proliferative effect on (an) immune cell(s), and/or    suppress/reduce the effector function(s) of (an) immune cell(s);-   (ii) are capable to inhibit, decrease or block immune cell    proliferation (without accumulation of dead cells);-   (iii) prevent (the onset of) activation and/or proliferation of    immune cells;-   (iv) lead to an inhibition, decrease or block of proliferating    immune cells (without accumulation of dead cells);-   (v) are capable of triggering the resting of (viable) immune cells    (without accumulation of dead cells);-   (vi) have a cytostatic effect on proliferating immune cells,    preferably lacking a cytotoxic effect;-   (vii) reduce or suppress an over-activity of immune cells;-   (viii) are capable to suppress/reduce secretion/production of    cytokines, in particular of IL-2, IFN-gamma and/or TNF-alpha;-   (ix) are capable to suppress/reduce degranulation/cytotoxicity of    PBMCs, in particular of CD107a⁺ CD8⁺ PBMCs;-   (x) are capable to suppress/reduce expression of IL-2 surface    receptor CD25 (on PBMCs);-   (xi) are capable to act in a similar manner as Cyclosporine A,    Muromonab-CD3 and/or Basiliximab; and/or-   (xii) do not induce a change in Ca²⁺ signalling and/or do not    induce/increase Ca²⁺ release from (animal) cells.

It is preferred that the herein defined cyclotide functions arefulfilled in the context of a cytostatic administration scheme. In thecontext of this administration scheme, the cyclotides, in particularkalata B1 or T20K, are capable to function without the accumulation ofdead cells, i.e. without a cytotoxic effect. This particularly appliesto the cyclotide functions as defined in sections (ii), (iv) and (v),supra.

The skilled person is readily in the position to test whether a givencyclotide or cyclotide mutant/variant can function in accordance withthe present invention, e.g. has one or more of the functions defined insections (i) to (xii), supra. For this purpose, the skilled person may,for example, rely on the assays described in the appended examples (e.g.examples 3 and 5, infra) and on respective assays for anti-proliferativeeffects as described in the art (Gruendemann, Journal ofEthnopharmacology 136, 3, SI, 2011, 444-451).

By relying on the herein described means and methods and his commongeneral knowledge, the skilled person is also in the position toidentify and isolate suitable cyclotides or cyclotide mutants/variants,for example in/from a (plant) extract. Hence, the skilled person isfurther able to identify and isolate not yet known cyclotides/cyclotidemutants/variants that can be used in accordance with the presentinvention. The use of such newly identified/isolated cyclotides inaccordance with the present invention is also envisaged herein.

In a preferred embodiment, the cyclotide to be used in accordance withthe present invention is a (naturally-occurring or native) non-graftedcyclotide, i.e. a cyclotide “per se” without any further(pharmaceutically) active compartments. It is known in the art thatcyclotides can act as scaffolds for other (pharmaceutically) activecompartments, like other therapeutic peptides (see, for example,Gunasehera, loc. cit.). Such grafted cyclotides, i.e. cyclotidescomprising a further (pharmaceutically) active compartment, are lesspreferred in the context of the present invention. In particular,grafted cyclotides are known to be cyclotides having at least onecomplete loop between two cysteine residues be replaced by a further(pharmaceutically) active compartment. This is to be seen in contrast tothe cyclotides and cyclotide mutants/variants to be preferably used inthe context of the present invention. Specifically, these cyclotidemutants/variants are mutated so that no further (pharmaceutically)active compartment is introduced. In principle, it is also possible withrespect to these peptide mutants/variants that one or more entire loopsbetween two cysteine residues are replaced by (a stretch of) furtheramino acid residues, as long as no further (pharmaceutically) activecompartment is introduced. The skilled person is readily in the positionto distinguish between a grafted cyclotide and a non-grafted cyclotideor a grafted and non-crafted cyclotide mutant/variant.

It is preferred that the immune cells referred to in items (i) to (xii),supra, but also the immune cells referred to herein elsewhere, areprimary immune cells.

Furthermore, it is preferred that the immune cells referred to in items(i) to (xii), supra, but also the immune cells referred to hereinelsewhere, are activated and/or proliferating immune cells. Alsopreferred is that the (primary) (activated and/or proliferating) immunecells are of human origin, i.e. are human (primary) (activated and/orproliferating) immune cells. Particular examples of immune cellsreferred to herein are (primary, activated and/or proliferating) PBMCsand lymphocytes, preferably T-Iymphocytes. Again, it is preferred thatthese PBMCs and (T-)lymphycytes are of human origin, i.e. human PBMCsand human (T-)lymphocytes. In one particular aspect, the PBMCs areCD107a⁺ CD8⁺ PBMCs.

In a further aspect, the present invention also relates to the use of anucleic acid molecule comprising a nucleotide sequence encoding theamino acid backbone/primary amino acid sequence of a cyclotide asdisclosed in context of this invention. For example, such nucleic acidmolecule may comprise a nucleotide sequence as depicted in any one ofSEQ ID NOs. 11, 12, 15 and 16 or a nucleotide sequence as comprised inany one of SEQ ID NOs. 11, 12, 15 and 16 and corresponding to the maturecyclotide or a nucleotide sequence which differs therefrom due to thedegeneracy of the genetic code.

The meanings of the terms “nucleic acid molecule(s)”, “nucleic acidsequence(s)” and “nucleotide sequence(s)” and the like are well known inthe art and are used accordingly in context of the present invention.

For example, when used throughout this invention, these terms refer toall forms of naturally-occurring or recombinantly generated types ofnucleotide sequences and/or nucleic acid sequences/molecules as well asto chemically synthesized nucleotide sequences and/or nucleic acidsequences/molecules. These terms also encompass nucleic acid analoguesand nucleic acid derivatives such as e.g. locked DNA, PNA,oligonucleotide thiophosphates and substituted ribo-oligonucleotides.Furthermore, these terms also refer to any molecule that comprisesnucleotides or nucleotide analogues.

Preferably, the terms “nucleic acid molecule(s)”, “nucleic acidsequence(s)” and “nucleotide sequence(s)” and the like refer todeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The “nucleic acidmolecule(s)”, “nucleic acid sequence(s)” and “nucleotide sequence(s)”may be made by synthetic chemical methodology known to one of ordinaryskill in the art, or by the use of recombinant technology, or may beisolated from natural sources, or by a combination thereof. The DNA andRNA may optionally comprise unnatural nucleotides and may be single ordouble stranded. “Nucleic acid molecule(s)”, “nucleic acid sequence(s)”and “nucleotide sequence(s)” also refer to sense and anti-sense DNA andRNA, that is, a nucleotide sequence which is complementary to a specificsequence of nucleotides in DNA and/or RNA.

Furthermore, the terms “nucleic acid molecule(s)”, “nucleic acidsequence(s)” and “nucleotide sequence(s)” and the like may refer to DNAor RNA or hybrids thereof or any modification thereof that is known inthe state of the art (see, e.g., U.S. Pat. Nos. 5,525,711, 4,711,955,5,792,608 or EP 302175 for examples of modifications). These moleculesof the invention may be single- or double-stranded, linear or circular,natural or synthetic, and without any size limitation. For instance, the“nucleic acid molecule(s)”, “nucleic acid sequence(s)” and/or“nucleotide sequence(s)” may be genomic DNA, cDNA, mRNA, antisense RNA,ribozymal or a DNA encoding such RNAs or chimeroplasts (Cole-StraussScience 1996 273(5280) 1386-9). They may be in the form of a plasmid orof viral DNA or RNA. “Nucleic acid molecule(s)”, “nucleic acidsequence(s)” and “nucleotide sequence(s)” and the like may also refer to(an) oligonucleotide(s), wherein any of the state of the artmodifications such as phosphothioates or peptide nucleic acids (PNA) areincluded.

The nucleic acid molecules as provided herein are particularly usefulfor producing a cyclic peptide of the invention, for example by acorresponding method disclosed herein.

The nucleic acid molecule as disclosed herein and described herein maybe comprised in a vector.

Said vector may be a cloning vector or an expression vector, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host/cells. The herein disclosed nucleic acid molecule maybe joined to a particular vector containing selectable markers forpropagation in a host. Generally, a plasmid vector is introduced in aprecipitate, such as a calcium phosphate precipitate or rubidiumchloride precipitate, or in a complex with a charged lipid or incarbon-based clusters, such as fullerens. Should the vector be a virus,it may be packaged in vitro using an appropriate packaging cell lineprior to application to host cells.

Preferably, the disclosed nucleic acid molecule is operatively linked toexpression control sequences (e.g. within the herein disclosed vector)allowing expression in prokaryotic or eukaryotic cells or isolatedfractions thereof. Expression of said polynucleotide comprisestranscription of the nucleic acid molecule, preferably into atranslatable mRNA. Regulatory elements ensuring expression in eukaryoticcells, preferably mammalian cells, are well known to those skilled inthe art. They usually comprise regulatory sequences ensuring initiationof transcription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript. Additional regulatoryelements may include transcriptional as well as translational enhancers.Possible regulatory elements permitting expression in prokaryotic hostcells comprise, e.g., the lac, trp or tac promoter in E. coli, andexamples for regulatory elements permitting expression in eukaryotichost cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-,RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or aglobin intron in mammalian and other animal cells. Beside elements whichare responsible for the initiation of transcription such regulatoryelements may also comprise transcription termination signals, such asthe SV40-poly-A site or the tk-poly-A site, downstream of thepolynucleotide. In this context, suitable expression vectors are knownin the art such as Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pSPORT1 (GIBCOBRL). Preferably, said vector is an expression vector and/or a genetransfer vector. Expression vectors derived from viruses such asretroviruses, adenoviruses, vaccinia virus, adeno-associated virus,herpes viruses, or bovine papilloma virus, may be used for delivery ofthe polynucleotides or vector of the invention into a targeted cellpopulation. Methods which are well known to those skilled in the art canbe used to construct a vector in accordance with this invention; see,for example, the techniques described in Sambrook, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. andAusubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994). Alternatively, thedisclosed polynucleotides and vectors can be reconstituted intoliposomes for delivery to target cells.

The term “isolated fractions thereof” refers to fractions of eukaryoticor prokaryotic cells or tissues which are capable of transcribing ortranscribing and translating RNA from the vector. Said fractionscomprise proteins which are required for transcription of RNA ortranscription of RNA and translation of said RNA into a polypeptide.Said isolated fractions may be, e.g., nuclear and cytoplasmic fractionsof eukaryotic cells such as of reticulocytes. Kits for transcribing andtranslating RNA which encompass the said isolated fractions of cells ortissues are commercially available, e.g., as TNT reticulolysate(Promega).

Again, like the disclosed nucleic acid molecules, also the disclosedvectors are particularly useful for producing a cyclic peptide of theinvention, for example by a corresponding method disclosed herein.

In a further aspect, disclosed herein is a recombinant host cellcomprising the nucleic acid molecule and/or the vector as disclosedherein. In context of this aspect, the nucleic acid molecule and/or thevector can, inter alia, be used for genetically engineering host cells,e.g., in order to express and isolate the amino acid backbone/primaryamino acid sequence of the cyclotides disclosed herein.

Said host cell may be a prokaryotic or eukaryotic cell; see supra. Thenucleic acid molecule or vector which is present in the host cell mayeither be integrated into the genome of the host cell or it may bemaintained extra chromosomally.

The host cell can be any prokaryotic or eukaryotic cell, such as abacterial, insect, fungal, plant, animal, mammalian or, preferably,human cell. Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae, orthose belonging to the group of hyphal fungi, for example severalpenicillia or aspergilla strains. The term “prokaryotic” is meant toinclude all bacteria which can be transformed or transfected with anucleic acid molecule for the expression of an amino acidbackbone/primary amino acid sequence of the cyclotides disclosed herein.Prokaryotic hosts may include gram negative as well as gram positivebacteria such as, for example, E. coli, S. typhimurium, Serratiamarcescens and Bacillus subtilis. A nucleic acid molecule coding for anamino acid backbone/primary amino acid sequence of the cyclic cyclotidesdisclosed herein can be used to transform or transfect a host using anyof the techniques commonly known to those of ordinary skill in the art.Methods for preparing fused, operably linked genes and expressing themin bacteria or animal cells are well-known in the art (Sambrook, supra).The genetic constructs and methods described therein can be utilized forexpression of the above mentioned amino acid backbone/primary amino acidsequence in, for example, prokaryotic hosts.

In general, expression vectors containing promoter sequences whichfacilitate the efficient transcription of the inserted polynucleotideare used in connection with the host. The expression vector typicallycontains an origin of replication, a promoter, and a terminator, as wellas specific genes which are capable of providing phenotypic selection ofthe transformed cells. The transformed prokaryotic hosts can be grown infermentors and cultured according to techniques known in the art toachieve optimal cell growth. The expressed peptides can then be isolatedfrom the grown medium, cellular lysates, or cellular membrane fractions.The isolation and purification of the microbially or otherwise expressedpeptides may be by any conventional means such as, for example,preparative chromatographic separations and immunological separationssuch as those involving the use of monoclonal or polyclonal antibodies(Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994)).

Again, like the nucleic acid molecules and the vectors as disclosed anddescribed herein, also the corresponding host cells are particularlyuseful for producing a cyclotide as disclosed herein, for example by thecorresponding method disclosed herein.

The skilled person is readily able to provide, i.e. synthesize, thecyclotides to be used in accordance with the present invention orisolate them from, for example, extracts (for example biologicalextracts like plant, fungal, animal or microbial extracts).

In particular, (bio-)chemically synthesizing approaches or generation ofcyclotides via recombination techniques may be employed. For example, amethod for producing a cyclotide may comprise the steps of

-   a) (i) culturing the herein disclosed recombinant host cell under    conditions such that the amino acid backbone of the herein disclosed    cyclotide is expressed, and recovering said amino acid backbone; or-    (ii) chemically synthesizing the amino acid backbone of the herein    disclosed cyclotide; and-   b) cyclization of said amino acid backbone to form the herein    disclosed cyclotide.

As mentioned above, the linear peptides/amino acid backbones of thecyclotides to be produced can also be produced by recombinantengineering techniques. Such techniques are well known in the art (e. g.Sambrook, supra). As also mentioned above, by this kind of production ofsaid linear peptides/amino acid backbones particular advantage can betaken of the herein disclosed and described nucleic acid molecules,vectors and/or host cells. The definitions correspondingly given aboveapply here, mutatis mutandis.

Several approaches of peptide synthesis particular synthesis approachesof cyclic peptides are known in the art. (e.g. Williams, ChemicalApproaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton:Chemistry of Peptide Synthesis. CRC-Press, 2005). The skilled person isreadily in the position to apply the prior art knowledge to theparticular requirements of the disclosed method for producing cyclicpeptides, based on the herein provided teaching.

This invention also relates to the use of a cyclotide obtainable orobtained by the above described approaches or method(s) in accordancewith the herein provided disclosure.

Terms like “immunosuppression”, “suppression of the immune system” and“suppression/reduction of the activation or efficacy of the immunesystem” are used herein in a comparable manner. The correspondingmeaning is known in the art and the terms are correspondingly usedherein. In particular, these terms refer to the suppression or decreaseof (a) parameter(s) of the immune system like, for example, (activatedand/or proliferating) (an) immune cell(s) as defined herein above.

In accordance with the present invention, (a) parameter(s) of the immunesystem may be selected from the group consisting of a

-   (i) immune cells (in particular those defined herein above), in    particular PBMCs, more particular lymphocytes, even more particular    T-lymphocytes;-   (ii) (a) effector function(s) of immune cells (in particular of    those defined herein above);-   (iii) cytokines, in particular the level, secretion and/or    production thereof;-   (iv) degranulation/cytotoxicity of immune cells, in particular of    CD107a⁺ CD8⁺ PBMCs; and-   (v) expression of (a) cytokine surface receptor (for example, IL-2    surface receptor CD25), in particular on PBMCs.

Cytokines in accordance with the present invention may be IL-2,IFN-gamma and TNF-alpha, whereby IL-2 is preferred.

“Suppression” or “reduction” in context of the present inventionparticularly means that the (defence) response of the immune systemagainst a(n) antigen(s)/(a) pathogen(s) is reduced. In the context ofthe present invention, this is not only to be seen with respect to anactivated, i.e. diseased state, of the immune system but also withrespect to the non-activated, i.e. normal, healthy state of the immunesystem. In this context it is clear that even in the normal, healthystate the immune system has a basic level of activation due to thecommon baseline of antigen/pathogen impact.

Hence, in one embodiment, the “suppression” of the immune system startsfrom a normal, healthy state of the immune system and, in anotherembodiment, from an activated, diseased state of the immune system.

In particular, it is envisaged in the context of the present inventionthat the immune system, in particular one or more parameters thereof, issuppressed/reduced by at least 10%, preferably by at least 20%, morepreferably by at least 30%, even more preferably by at least 50%, evenmore preferably by at least 80%, even more preferably by at least 90%,even more preferably by at least 95%, even more preferably by at least99% and most preferably 100% of the initial status of the immune system(being either a diseased or a non-diseased status), in particular of oneor more parameters thereof. Herein, suppressing/reducing the immunesystem particularly means suppressing/reducing proliferation of immunecells. The skilled person is readily in the position to test the degreeof suppression of the immune system, for example by determining theproliferative activity of immune cells or the fraction ofproliferating/activated immune cells. Moreover, the skilled person isreadily in the position to determine for a givenimmunosuppressant/immunosuppressive drug the IC₅₀ for the respectiveimmunosuppressive effect/activity.

It is clear to the skilled person that, in accordance with the presentinvention, the disclosed pharmaceutical composition or cyclotide may beadministered in a pharmaceutically/therapeutically effective dose, whichmeans that a pharmaceutically/therapeutically effective amount of thecompound administered is reached. Preferably, apharmaceutically/therapeutically effective dose refers to that amount ofthe compound administered (active ingredient) that produces ameliorationof symptoms or a prolongation of survival of a subject which can bedetermined by the one skilled in the art doing routine testing.

It is of note that the dosage regimen of the compounds to beadministered in accordance with the present invention will be determinedby the attending physician and clinical factors. As is well known in themedical arts, that dosages for any one patient depends upon manyfactors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. A person skilled in the art is aware of and is able totest the relevant doses, the compounds to be medically applied inaccordance with the present invention are to be administered in.

It is particularly envisaged in the context of the present inventionthat the cyclotide is to be administered so that a cytostatic butlittle, preferably no, cytotoxic activity/effect occurs.

For this purpose, the cyclotide may, for example, be administered sothat a (serum) concentration in the range of 1 to 50 μM, preferably inthe range of 1 to 15 μM, more preferably in the range of 3 to 10 μM,more preferably in the range of 4 to 9 μM and even more preferably inthe range of 5 to 9 μM is reached. In particular, the cyclotide may beadministered via a particular route of administration and/or at anamount/dose to reach a (serum) concentration in the range of 1 to 50 μM,preferably in the range of 1 to 15 μM, more preferably in the range of 3to 10 μM, more preferably in the range of 4 to 9 μM and even morepreferably in the range of 5 to 9 μM.

Further, the cyclotide may, for example, be administered at a dose inthe range of 0.1 to 15 mg/kg, preferably in the range of 0.1 to 12mg/kg, more preferably in the range of 1 to 12 mg/kg, more preferably inthe range of 1 to 10 mg/kg, more preferably in the range of 5 to 10mg/kg, and even more preferably at a dose of about 10 mg/kg.

The dose may be administered on a daily, monthly or, preferably, weeklybasis. The cyclotide may be administered in form of 1 or more singledoses; in particular, 1, 2, 3, 4 or 5 single doses. The cyclotide may,for example, be administered intravenously or intraperitoneally. Nonlimiting Examples of particular administration schemes are 3 singleintravenous injections of about 10 mg/kg at weekly intervals or a singleintraperitoneal dose of about 10 mg/kg. Further possible administrationschemes are describe herein below.

The skilled person is readily in the position to find out the particularroute of administration and amount/dose of a given cyclotide to beapplied in order to reach cytostatic but little/no cytotoxic activity.

In one specific embodiment, the herein described pharmaceuticalcomposition may further comprise one or more additionalimmunosuppressant(s). Preferably, this (these) additionalimmunosuppressant(s) is (are) not a part of a grafted form of thecyclotide but is independently comprised in the pharmaceuticalcomposition. In another specific embodiment, the additionalimmunosuppressant(s) is (are) administered separately. In anotherspecific embodiment, the herein described cyclotide may be administeredtogether with one or more additional immunosuppressant(s), i.e. prior,simultaneously or subsequently with respect to the additionalimmunosuppressant(s). Non-limiting examples of an additionalimmunosuppressant may be selected from the group consisting ofCyclosporine A, Muromonab-CD3 (Orthoclone OKT3®) andBasiliximab)(Simulect®).

The herein described pharmaceutical composition may also comprise one ormore (anti-immune cell-proliferative) cyclotides. Hence, in a furtherspecific embodiment, the herein described pharmaceutical composition maycomprise at least two, three, four or five cyclotides as describedherein. In a further specific embodiment, one of the herein describedcyclotides is to be administered together with, i.e. prior to,simultaneously with or subsequently to another, different, of the hereindescribed cyclotides.

In one embodiment, the pharmaceutical composition of the presentinvention may comprise, or be in form of, an (native) extract, inparticular a (native) plant extract.

Non-limiting examples of plants from which such an extract may beobtained are Betula pendula, Oldenlandia affinis, plants from theViolaceae family (e.g. Viola sp., preferably V.odorata and V. tricolor),Squash species (Cucurbitaceae family), Ecballium species, legume species(Fabaceae family) and Psychotria species (Rubiaceae family; for examplePsychotria polyphlebia, P. poeppigiana, P. chiapensis, P. borucana, P.buchtienii, P. pillosa, P. mortomiana, P. deflexa, P. makrophylla, P.elata, P. solitudinum, P. capitata).

As mentioned above, one embodiment of the present invention relates to(a pharmaceutical composition comprising) a cyclotide for use in immunesuppression or to a method for immune suppression by administering (apharmaceutical composition comprising) a cyclotide. In anotherembodiment, the present invention relates to (a pharmaceuticalcomposition comprising) a cyclotide for use in treating or preventing adisease or disorder and a method of treating or preventing a disease ordisorder, said disease or disorder is caused by the activity of theimmune system, i.e. a disease or disorder which can be treated,prevented or ameliorated by immunosuppression. In this context, not onlythe suppression of an over-active immune system to a lower level, forexample a normal, non-diseased level, is envisaged, but also thesuppression of a normal, healthy-state immune system is envisaged. Thelatter is, for example, particularly relevant with respect to organtransplantation approaches. The skilled person knows, or at least cantest for, particular diseases which can be treated or prevented bysuppressing the immune system. Examples of such diseases or disordersare given in Kumar (“Clinical Medicine”, 3^(rd) edition(1994), BaillièreTindall).

In particular, the disease or disorder to be treated or prevented inaccordance with this invention is selected from the group consisting of:

-   (i) autoimmune disorders;-   (ii) hypersensitivity disorders; and-   (iii) immune cell-mediated inflammations.

The meaning and scope of “autoimmune disorder”, “hypersensitivitydisorder” and “immune cell-mediated inflammation” is known in the artand can, for example, be deduced from Kumar (“Clinical Medicine”, 3^(rd)edition, 1994, Baillière Tindall).

Particular examples of autoimmune disorders to be treated or preventedare selected from the group consisting of:

-   (i) Multiple Sclerosis;-   (ii) Psoriasis;-   (iii) Systemic Lupus Erythematosus;-   (iv) Sjögren's syndrome;-   (v) Rheumatoid Arthritis (RA), in particular severe RA;-   (vi) Idiopathic Thrombocytopenic Purpura;-   (vii) Diabetes;-   (viii) Vasculitis; and-   (ix) Crohn's disease.

Particular examples of hypersensitivity disorders to be treated orprevented are graft-versus-host disorders and Contact Dermatitis.

A particular example of an immune cell-mediated inflammation is alymphocyte-mediated inflammation, in particular a T-cell-mediatedinflammation. Particular examples of lymphocyte-mediated inflammationsto be treated or prevented are Keratoconjunctivitis sicca and Dry EyeSyndrome (DES). Corneal clarity is required for optimal vision and canbe affected severely by any form of corneal inflammation. This ismediated by infiltrating leukocytes and pathological blood vesselformation in the long-run. In general, any occurring cornealinflammation is to be treated especially if the central cornea isinvolved. Once a corneal scar established, keratoplasty becomesnecessary to restore corneal transparency that is indispensable foroptimal vision.

In one embodiment, it is particularly envisaged that diseases ordisorders of a sub-group of the above (or herein elsewhere) defineddiseases or disorders are to be treated/prevented, said sub-group ofdiseases or disorders comprises those diseases or disorders which

-   (i) come along with and/or are caused by an (over-)activated immune    system and/or (over-)activated/increased parameter(s) of the immune    system or-   (ii) which can be treated/prevented by suppressing the immune system    (starting from an (over-)activated, diseased state or from a normal,    healthy state). Within this sub-group, particularly those    diseases/disorders are to be treated/prevented which come along with    and/or are caused by (over-)activated immune cells or which can be    treated/prevented by reducing the (proliferating) activity of immune    cells.

What has been said with respect to the meaning of “immune cells” and“parameter(s) of the immune system” herein elsewhere also applies here,mutatis mutandis.

In another embodiment, immune cells, in particular proliferation of thesame, are/is to be suppressed in the context of the treatment/preventionof this invention. Preferably, such immune cells are (primary) activated(T-)lymphocytes and/or peripheral blood mononuclear cells (PBMC). Again,what has been said with respect to the meaning of “immune cells” hereinelsewhere also applies here mutatis mutandis.

In another embodiment, (a) parameter(s) of the immune system are/is tobe suppressed/reduced in the context of the treatment/prevention of thisinvention. What has been said with respect to the meaning of“parameter(s) of the immune system” herein elsewhere also applies here,mutatis mutandis.

In one specific aspect, the disease or disorder to be treated/preventedin accordance with this invention is a disease or disorder mediated by acytokine pathway, in particular the IL-2 pathway (via CD25).

In another specific aspect, the disease or disorder to betreated/prevented is a disease or disorder

-   (i) which cannot be treated or is not to be treated by an induction    or increase of Ca²⁺ release; and/or-   (ii) which does not come along or is not related to a change in Ca²⁺    signalling.

In another specific embodiment, the disease to be treated/prevented isnot a disease that can be treated/prevented by inhibiting the activityof tryptase, i.e. is a tryptase-independent disease or disorder.

Each or more of the above embodiments/aspects particularly applies/applyto the above (or herein elsewhere) defined or exemplified diseases ordisorders, in particular to the diseases or disorders as defined orexemplified in sections (i) to (iii) or (i) to (ix), supra. Moreparticular, each or more of the above embodiments/aspects applies/applyto a sub-group of these diseases or disorders.

Beside their amino acid backbone, the cyclotides to be used inaccordance with the invention may further comprise (e.g. have covalentlybound) (a) further substituent(s), like labels, anchors (likeproteinaceous membrane anchors), tags (like HIS tags). Thesubstituent(s) can be bound covalently or non-covalently to thecyclotides and directly or via linkers. The skilled person is readily inthe position to find out appropriate linkers to be employed in thiscontext. Moreover, appropriate substituents and methods for adding themto a cyclotide are known to those of ordinary skill in the art.

Examples of labels include, inter alia, fluorochromes (like fluorine-18,fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radishperoxidase, β-galactosidase, alkaline phosphatase), radio/radioactiveisotopes (like 32P, 33P, 35S, 125I or 123I, 135I, 124I, 11C, 15O),biotin, digoxygenin, colloidal metals, chemi- or bioluminescentcompounds (like dioxetanes, luminol or acridiniums). One non-limitingexample of a label that may be bound to the cyclotide is a fluorochrome,like a FRET fluorochrome, for example a GFP, YFP or CFP variant (e.g.GFP, YFP, CFP, eGFP, EYFP or ECFP). A variety of techniques areavailable for labeling biomolecules, and comprise, inter alia, covalentcoupling of enzymes or biotinyl groups, phosphorylations,biotinylations, random priming, nick-translations, tailing (usingterminal transferases). Such techniques are, e.g., described in Tijssen,“Practice and theory of enzyme immunoassays”, Burden and von Knippenburg(Eds), Volume 15 (1985); “Basic methods in molecular biology”, Davis LG, Dibmer M D, Battey Elsevier (1990); Mayer, (Eds) “Immunochemicalmethods in cell and molecular biology” Academic Press, London (1987); orin the series “Methods in Enzymology”, Academic Press, Inc.Corresponding detection methods comprise, but are not limited to,autoradiography, fluorescence microscopy, direct and indirect enzymaticreactions, etc.

The cyclotides as described and defined herein, in particular theabove-described labelled cyclotides may be employed in biodistributionstudies, i.e. studies resulting in a pattern of distribution of thecyclotide, for example in an animal or, preferably a humansubject/patient. For example, such biodistribution studies may compriseimaging by single-photon or PET imaging devices.

Administration of the pharmaceutical composition or the cyclotide(s) inaccordance with this invention may be effected by different ways. Suchmay be, for example, oral, intravenous, intraarterial, intraperitoneal,intravesical or subcutaneous administrations or administration byinhalation as well as transdermal administration. Other examples areparenteral, such as subcutaneous, intravenous, intramuscular,intraperitoneal, intrathecal, transdermal, transmucosal, transpulmonalsubdural administrations, local or topical administrations andadministrations via iontopheresis, sublingual administrations,administrations by inhalation spray or aerosol or rectaladministrations, and the like.

In particular, for patients and/or for particular medical uses,particular administration routes like blood infusion (e.g. intravenousinfusion), rectal administration (e.g. in form of enemas orsuppositories) or topical administration routes (in particular when eyediseases like the dry eye syndrome are to be treated) may be indicated.

A carrier optionally comprised in the pharmaceutical composition of theinvention or to be administered together with the pharmaceuticalcomposition or the cyclotide of the invention may particularly be apharmaceutically acceptable carrier, excipient or diluent.

Such carriers are well known in the art. The skilled person is readilyin the position to find out such carriers which are particularlysuitable to be employed in accordance with the present invention.

Pharmaceutically acceptable carriers/excipients that may be used in theformulation of the pharmaceutical compositions comprising the activecompounds as defined herein (or a salt thereof) may generally comprisecarriers, vehicles, diluents, solvents such as monohydric alcohols suchas ethanol, isopropanol and polyhydric alcohols such as glycols andedible oils such as soybean oil, coconut oil, olive oil, safflower oilcottonseed oil, oily esters such as ethyl oleate, isopropyl myristate;binders, adjuvants, solubilizers, thickening agents, stabilizers,disintergrants, glidants, lubricating agents, buffering agents,emulsifiers, wetting agents, suspending agents, sweetening agents,colourants, flavours, coating agents, preservatives, antioxidants,processing agents, drug delivery modifiers and enhancers such as calciumphosphate, magnesium state, talc, monosaccharides, disaccharides,starch, gelatine, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-R-cyclodextrin, polyvinylpyrrolidone,low melting waxes, ion exchange resins. Other suitable pharmaceuticallyacceptable carriers/excipients are described in Remington'sPharmaceutical Sciences, 15^(th) Ed., Mack Publishing Co., New Jersey(1991). In the following, several non-limiting administration schemesand the use of correspondingly suitable pharmaceutically acceptablecarrier are described.

For an administration of the pharmaceutical composition or thecyclotides in accordance with this invention via subcutaneous (s.c.) orintravenous (i.v.)/intraarterial (i.a.) injection, cyclotides (orencoding sequences) may be formulated in aqueous solution, preferably inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiologically saline buffer. For transmucosal andtranspulmonal administration, penetrants appropriate to the barrier tobe permeated are used in the formulation. Such penetrants are generallyknown in the art.

The use of pharmaceutical acceptable carriers to formulate thecyclotides into dosages or pharmaceutical compositions suitable forsystemic, i.e. intravenous/intraarterial, or subcutaneous administrationis within the scope of the present invention. With proper choice ofcarrier and suitable manufacturing practice, the compositions of thepresent invention, in particular those formulated as solutions, may beadministered parenterally, such as by intravenous injection. Thecompounds can be readily formulated using pharmaceutically acceptablecarriers well known in the art into dosages suitable for subcutaneous ororal administration. Such carriers enable the compounds according to thepresent invention to be formulated as tablets, pills, capsules, dragees,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated.

Compounds according to the present invention, or medicaments orpharmaceutical compositions comprising them, intended to be administeredintracorporally/intracellularly may be administered using techniqueswell known to those of ordinary skill in the art. For example, suchagents may be encapsulated into liposomes, then administered asdescribed above. Liposomes are spherical lipid bilayers with aqueousinteriors. All molecules present in an aqueous solution at the time ofliposome formation are incorporated into the aqueous interior. Theliposomal contents are both protected from the external microenvironmentand, because liposomes fuse with cell membranes, are efficientlydelivered near the cell surface. Delivery systems involving liposomesare disclosed in U.S. Pat. No. 4,880,635 to Janoff et al. Thepublications and patents provide useful descriptions of techniques forliposome drug delivery.

Pharmaceutical compositions comprising a compound according to thepresent invention for parenteral and/or subcutaneous administrationinclude aqueous solutions of the active compound(s) in water-solubleform. Additionally, suspensions of the active compounds may be preparedas appropriate oily injection suspensions. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil or castor oil, orsynthetic fatty acid esters, such as ethyl oleate or triglycerides, orliposomes. Aqueous injections suspensions may contain compounds whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, or the like. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions and to allow for a constantly slow release of thesubstance in the organism.

A “patient”/“subject” for the purposes of the present invention, i. e.to whom a pharmaceutical composition or cyclotide according to thepresent invention is to be administered or who suffers from the diseasea disorder as defined and described herein, includes both humans andanimals and other organisms. Thus the compositions and methods of thisinvention are applicable to or in connection with both, human therapyand veterinary applications including treating/preventing procedures andmethods. In the preferred embodiment the patient/subject is a mammal,and in the most preferred embodiment the patient/subject is human.

The present invention further relates to a method of screening forand/or selecting an immunosuppressive cyclotide comprising the step of

-   i) contacting a cyclotide or a (plant) extract containing a    cyclotide with (a sample of) an (activated) cell of the immune    system and determining the proliferative activity of said cell,-    wherein a suppressed or reduced proliferative activity (as compared    to a control) is indicative for the immunosuppressive activity of    the cyclotide; or-   ii) administering to an animal model a pharmaceutically effective    amount of a cyclotide or a (plant) extract containing a cyclotide    and determining ((a) parameter(s) of) the immune system or (a)    clinical sign(s)/the presence of a disease or disorder as defined    herein,-    wherein the suppression or reduction of (the parameter(s) of) the    immune system or the decrease of the clinical sign(s)/amelioration    of the disease or disorder (as compared to a control) is indicative    for the immunosuppressive activity of the cyclotide.

The method of screening for and/or selecting may further comprise thestep of isolating and/or identifying the immunosuppressive cyclotide(from/in the (plant) extract). For example, said step of isolatingand/or identifying may comprise (nano) LC-MS/MS or LC-MS reconstruction,preferably a combination of both, (nano) LC-MS/MS and LC-MSreconstruction. Optionally, said step may further comprise (automated)database searching and/or manual de novo peptide sequencing by assigningb- and y-fragment ions from MS/MS spectra.

Moreover, the method of screening for and/or selecting may furthercomprise a step of determining the biodistribution pattern of the(isolated and/or identified) immunosuppressive cyclotide in (a sampleof) a human or animal subject. Examples of corresponding biodistributiontechniques are described herein above.

A suitable control as to the herein disclosed method of screening forand/or selecting an immunosuppressive cyclotide may be (a sample of) an(activated) cell of the immune system that

-   (i) has not been contacted with the cyclotide or the (plant) extract    containing a cyclotide; or-   (ii) has been contacted with a cyclotide not having    immunosuppressive activity.

Another suitable control as to the herein disclosed method of screeningfor and/or selecting an immunosuppressive cyclotide may be an animalmodel to which

-   (i) no such cyclotide or (plant) extract containing a cyclotide has    been administered; or-   (ii) a cyclotide not having immunosuppressive activity has been    administered (at a comparable or the same amount).

The present invention further relates to a method of screening forand/or selecting a mutation which, when introduced into a cyclotide,results in a mutated cyclotide having an induced or enhancedimmunosuppressive activity as compared to the non-mutated cyclotide,said method comprising the steps of

-   (i) introducing a mutation into a cyclotide; and-   (ii) contacting the so mutated cyclotide with (a sample of) an    (activated) cell of the immune system and determining the    proliferative activity of said cell, wherein a reduced proliferative    activity as compared to a control indicates that the mutation    confers (enhanced) immunosuppressive activity to the cyclotide; or    administering to an animal model a pharmaceutically effective amount    of the so mutated cyclotide and determining ((a) parameter(s) of)    the immune system or (a) clinical sign(s)/the presence of a disease    or disorder as defined herein, wherein the suppression or reduction    of (the parameter(s) of) the immune system or the decrease of the    clinical sign(s)/amelioration of the condition/disorder as compared    to a control indicates that the mutation confers (enhanced)    immunosuppressive activity to the cyclotide.

A suitable control as to the herein disclosed method of screening forand/or selecting a mutation may be (a sample of) an (activated) cell ofthe immune system that

-   (i) has not been contacted with the mutated cyclotide; or-   (ii) has been contacted with the non-mutated form of the cyclotide    or with a cyclotide not having immunosuppressive activity.

Another suitable control as to the herein disclosed method of screeningfor and/or selecting a mutation may be an animal model to which

-   (i) no such mutated cyclotide has been administered; or-   (ii) the non-mutated form of the cyclotide or a cyclotide not having    immunosuppressive activity has been administered

Non-limiting examples of cyclotides not having immunosuppressiveactivity are selected from the group consisting of the Kalata B1 mutantsT8K, V10A and V10K as disclosed herein.

Suppression or reduction of the proliferative activity, (theparameter(s) of) the immune system or the decrease of the clinicalsign(s)/amelioration of the condition/disorder as compared to a controlpreferably means a suppression or reduction by at least 20%, morepreferably by at least 30%, more preferably by at least 40%, morepreferably by at least 50%, more preferably by at least 60%, morepreferably by at least 70%, more preferably by at least 80%, morepreferably by at least 90% and more preferably by at least 95% ascompared to a control.

The present invention further relates to a method of producing animmunosuppressive cyclotide comprising the step of introducing amutation screened for and/or selected according to the above method intoa cyclotide.

In general, “mutation” in the context of the present invention means anychange in the structure of the (native or wildtype) cyclotide, inparticular in the primary amino acid sequence thereof. More particular,“mutation” means that one or more amino acid residues of the (native orwildtype) cyclotide are replaced, substituted or added. In one specificaspect, “mutation” refers to a point mutation, i.e. to the replacement,substitution or addition of one amino acid residue. In a more specificaspect, “mutation” refers to the replacement of one amino acid residue.What has been said with respect to the mutated/variant forms to be usedin accordance with the present invention herein elsewhere also appliesto the meaning of the term “mutation”, mutatis mutandis.

If not specified differently, “induction”, “induce” or “induced” in thecontext of this invention means starting from a baseline which isvirtually zero. “Increase”/“increased” or “enhance”/“enhanced” notnecessarily means starting from a baseline which is virtually zero butmay also mean starting from a level which is already above zero. Forexample, an induced immunosuppressive activity is meant, when thereinitially was no immunosuppressive activity at all; anenhanced/increased immunosuppressive activity is meant, when thereinitially was already some immunosuppressive activity which is thenfurther enhanced/increased.

A preferred animal model to be applied in the context of the hereindisclosed method of screening for and/or selecting is an animal modelfor any one of the diseases or disorders as defined herein.Corresponding animal models are known in the art. For example, such ananimal model may be an animal model for an autoimmune disease, like, forexample, MS. A non-limiting example for an MS animal model is an EAEanimal model, for example an EAE mouse model. Another example of ananimal model which may be applied in this context is a (mouse, rat, andthe like) model for dry eye syndrome (DES), like, for example, a modelfor DES using Fisher and/or Lewis rats.

The use of such animal models in the context of the screening/selectionmethods of the invention is described in more detail in the followingnon-limiting example. Corneal clarity is required for optimal vision andcan be affected severely by any form of corneal inflammation. This ismediated by infiltrating leukocytes and pathological blood vesselformation in the long-run. Independently of the reasons, any occurringcorneal inflammation has to be treated especially if the central corneais involved. Once a corneal scar established, keratoplasty becomesnecessary to restore corneal transparency that is indispensable foroptimal vision. Orthotopic corneal transplantation may be performedbetween Fisher and Lewis rats. Recipient rats may either be treated witha control or the component (i.e. cyclotide) of interest. The therapy maybe administered intraperitoneally (or another method described above)for, for example, 14 days. All treatments may be controlled in asyngeneic setting. Corneal grafts may be fixed with eight interruptedsutures and protected by a blepharorraphy for the initial 3 days aftertransplantation. After removal of the blepharorraphy, the grafts may beexamined by two independent investigators for signs of opacity,vascularization, and edema according to an internationally acceptedscore. Opacification of the graft may be scored as follows: 0=noopacity; 1=slight opacity, details of iris clearly visible; 2=moderateopacity, some details of iris no longer visible; 3=strong opacity, pupilstill recognizable; 4=total opacity. Rejection may be defined ascomplete graft opacification (grade 4). Additionally, all animals may bemonitored daily for signs of toxic side effects, such as weight loss.Rats may be sacrificed for histological characterization of theleukocytic infiltrate in the graft. Additionally, draining andnon-draining submandibular lymph nodes as well as spleen may be preparedfor flow cytometric analysis of T lymphocytes activation and T cellapoptosis. Finally, the systemically induced strength of the T cellresponse may be determined by a mixed-leukocyte reaction in both lymphnodes mentioned.

The skilled person is readily in the position to apply and adapt thedescribed exemplified use of an DES model also to other animal models ofany one of the diseases or disorders as defined herein.

In one specific aspect, (a sample of) a (activated) cell of the immunesystem may be seen as an “animal model” in accordance with the presentinvention.

It is particularly envisaged that the cyclotide to be screened/selectedis a cyclotide as defined herein. The respective definitions apply heremutatis mutandis.

In the context of the herein disclosed methods of screening/selecting,the (activated) cell or the parameter of the immune system may be animmune cell or parameter of the immune system as defined and describedherein elsewhere. The respective definitions also apply here, mutatismutandis.

A suitable “sample” in accordance with the present invention includes,but is not limited to, (a) biological or medical sample(s), like, e.g.(a) sample(s) comprising cell(s) or tissue(s). For example, such (a)sample(s) may comprise(s) biological material of biopsies. The meaningof “biopsies” is known in the art. For instance, biopsies comprisecell(s) or tissue(s) taken, e. g. by the attending physician, from apatient/subject as described herein. Exemplarily, but not limiting, thebiological or medical sample to be analysed in context of the presentinvention is or is derived from blood, plasma, white blood cells, urine,semen, sputum, cerebrospinal fluid, lymph or lymphatic tissues or cells,muscle cells, heart cells, cells from veins or arteries, nerve cells,cells from spinal cord, brain cells, liver cells, kidney cells, cellsfrom the intestinal tract, cells from the testis, cells from theurogenital tract, colon cells, skin, bone, bone marrow, placenta,amniotic fluid, hair, hair and/or follicles, stem cells (embryonic,neuronal, and/or others) or primary or immortalized cell lines(lymphocytes, macrophages, or cell lines). Preferred “samples” inaccordance with the present invention are those derived from blood orplasma. The biological or medical sample as defined herein may also beor be derived from biopsies, for example biopsies derived from hearttissue, veins or arteries.

In one aspect of the pharmaceutical composition or methods of thisinvention, the anti-proliferative effect or suppression/reduction ismediated in a cytokine-depending manner, for example in an IL-2-,IFN-gamma- and/or TNF-alpha-depending manner, and/or can be antagonizedby a cytokine, for example by IL-2.

The present invention further relates to a method of producing animmunosuppressive pharmaceutical composition comprising the step ofmixing

-   (i) a cyclotide as defined herein; or-   (ii) a cyclotide screened for, selected, produced, isolated or    identified as described herein    with a pharmaceutically acceptable carrier.

The present invention further relates to a mutated cyclotide havingimmunosuppressive activity and, in particular, to a mutated cyclotide asdefined and described herein (for example a mutated cyclotide comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:3 to 7 or a cyclotide consisting of a head-to-tail cyclized form of anamino acid sequence selected from the group consisting of SEQ ID NOs: 3to 7.

Furthermore, the present invention related to a pharmaceuticalcomposition comprising a mutated cyclotide having immunosuppressiveactivity and optionally a pharmaceutically acceptable carrier, excipientor diluent. Also in this context, it is particularly envisaged that themutated cyclotide is a mutated cyclotide as defined herein above.

The present invention is further described by reference to the followingnon-limiting figures and examples.

The Figures show:

FIG. 1. Structure and sequence diversity of cyclotides. The structure ofthe typical cyclotide kalata B1 is shown in black cartoon. The sixconserved cysteines are labeled with roman numerals and the resultingcysteine-knot disulfide connectivity (C_(I)-C_(IV), C_(II)-C_(V) andC_(III)-C_(VI)) is shown. The amino acid sequence and disulfideconnectivity of kalata B1 is shown below the structure cartoon. Thenumbers (n) indicate the possible length (in amino acids) of theinter-cysteine loops comprising all currently known cyclotides(according to Ireland et al. (Ireland, 2010, J Nat Prod, 73,1610-1622)). The inter-cysteine loops can tolerate a wide variety ofamino acid substitutions and are an indicator of the combinatorialdiversity of the cyclotide scaffold. The positions of syntheticmutations that have been introduded during this study are indicated byamino acid one-letter code, number and asterisk. The natural point ofcyclysation is indicated by an arrow.

FIG. 2. Effects of the O. affinis cyclotide extract on cellproliferation of activated human peripheral blood mononuclear cells.CFSE-labelled primary human PBMC were antibody-activated (anti-CD3/CD28mAbs) and cultured in the presence of medium (ctrl), camptothecin (CPT,30 μg/mL) or different concentrations (50-100 μg/mL) of O. affiniscyclotide extract. The cells were further analyzed for cell viabilityand proliferation capacity using flow forward-side-scatter-based flowcytometric analysis (A and B). Cell division analysis were assessed byFACS and illustrated as representative dot plots (C). Results aresummarized from three independent experiments in (D) and data arepresented as mean±SEM.

FIG. 3. Nano LC-MS chromatogram of O. affinis cyclotides. The nanoflowelution profile of cyclotides from O. affinis was monitored with UVabsorbance at 214 nm and mass spectrometry. The HPLC graph of arepresentative crude cyclotide extract is shown and its major cyclotidesare indicated by name and relative abundance. The relative cyclotidecontent (mean±SEM) was determined by peak integration of fiveindependent experiments (see Table 6). HPLC and MS conditions forcyclotide analysis are shown in the Methods Section.

FIG. 4. Effects of kalata B1 on cell proliferation of activated humanperipheral blood mononuclear cells. The influence of medium (ctrl),camptothecin (CPT, 30 μg/mL) or different concentrations of kalata B1(1.8-14 μM) on proliferation of CFSE⁺ anti-CD3/CD28 mAbs-activated humanprimary PBMC was measured by cell division analysis using flowcytometry. Data are presented as mean±SEM of four independentexperiments.

FIG. 5. Effects of kalata B1 on cytotoxicity of activated humanperipheral blood mononuclear cells. Human primary PBMC were activatedwith anti-CD3/CD28 mAbs in the presence of medium (ctrl), camptothecin(CPT, 30 μg/mL), Triton-X 100 (T-x) or different concentrations ofkalata B1 (1.8-14 μM) and analyzed for “subG1” DNA content (A) by flowcytometry. Cells were stained with annexin V and propidium iodide (PI)to assess the percentages of viable (annexin V⁻/PI⁻), apoptotic (annexinV⁺/PI⁻ or annexin V⁺/PI⁺) and necrotic (annexin V⁻/PI⁺) cells. Dot plotswere analyzed and representative graphs are shown in (B). Results fromthree independent experiments are summarized and data are presented asmean±SEM (C and D). n.d.=not detectable.

FIG. 6. Structural alignment of kalata B1 and B2. The NMR solutionstructures of kalata B1 (PDB code: 1NB1) and kalata B2 (1 PT4) werestructurally aligned using PyMol. Alignment of all atoms (A) results inan RMSD of 0.725 Å (only the five differing residues are highlighted inbold stick mode, the remaining residues are shown with thin lines) andthe backbone atoms (B) fit to a RMSD of 0.599 Å. The sequences of bothcyclotides are shown below the aligned structures with differingresidues indicated by black boxes.

FIG. 7. Determination of IC₅₀ for anti-proliferative effect of kalata B1on PBMC. The IC50 of the anti-proliferative effects of kalata B1 (seeFIG. 4) has been determined by non-linear regression analysis (loginhibitor vs. normalized response) using GraphPad Prism.

FIG. 8. Effects of melittin on cell proliferation and cytotoxicity ofactivated human peripheral blood mononuclear cells. Antibody(anti-CD3/CD28 mAbs)-activated human primary lymphocytes were culturedin the presence of medium (ctrl), camptothecin (CPT, 30 μg/mL), Triton-X100 (T-x) or different concentrations of melittin (0.05-1.6 μM) for flowcytometric analysis of cell division (A), “subG1” DNA content (B) orapoptotic (C) and necrotic (D) cell content. For apoptotic and necroticdetection cells were stained with annexin V and propidium iodide toassess the percentages of viable (annexin V−/PI−), apoptotic (annexinV+/PI− or annexin V+/PI+) and necrotic (annexin V−/PI+) cells. Data arepresented as mean±SEM of three to four independent experiments. n.d.=notdetectable.

FIG. 9. Effects of cyclotide mutants on cell proliferation of activatedhuman peripheral blood mononuclear cells (PBMC). The influence ofnon-activated (∅), medium (ctrl), camptothecin (CPT, 30 μM), cyclosporinA (CsA, 1 μg/mL) or different concentrations of cyclotides (1.8-14 μM)on proliferation of CFSE+ anti-CD3/CD28 (each 100 ng/mL) mAbs-activatedhuman primary PBMC was measured by cell division analysis using flowcytometry. Data are presented as mean+SD of at least two independentdonors and experiments. Cyclotide mutants G18K, N29K and T20K showanti-proliferative capacity. T20K+G1K is cytotoxic at 14 μM. Controlsare similar in each bar diagram. Results with CD3-purified cells are inagreement with those data (see Table 2).

FIG. 10. Activity of kalata B1 in vivo in experimental auto-immuneencephalomyelitis in mice. (A) Clinical score of EAE mice aftervaccination with kalata B1 (light line) or PBS control (black line) wasdetermined as outlined in Materials and Methods Section. Vaccinationwith the cyclotide resulted in a reduction in the incidence and severityof EAE. (B) The influence of kalata B1 vaccination on the formation ofCNS inflammatory and demyelinating lesions was examined by histologicalstudies of fixed tissue using haemotoxylin/eosin, Luxol fast blue andBielshowsky silver staining. The CNS of all mice treated with PBS showedinflammatory lesions, demyelination and axonal damage were particularlyflorid in the cerebellum and spinal cord (indicated by arrows).Vaccination with kalata B1 leads to a reduction of both clinical signsand histological lesions of EAE. (C) Proliferation of spleen cells inresponse to the encephalitogen MOG₃₅₋₅₅ and stimulation by thepolyclonal activators, anti-CD3 and anti-CD28 antibodies showsregardless of the treatment regimen, splenocytes from all vaccinatedmice proliferated to MOG and these splenocytes displayed strongproliferative responses to the anti-CD3/CD28 antibodies. (D) Suppressionof EAE by kalata B1 is not associated with a suppression of anti-MOGantibodies production. As shown, anti-MOG antibodies were detected inall sera regardless of the vaccination regimen. (E, F) MOG-reactive Tcells in protected animals did not switch to an anti-inflammatory T cellphenotype. Significantly reduced levels of the chemokine MIG (E) andTNFα (F) were demonstrated in non-stimulated spleen cell supernatantsgenerated from animals treated with kalata B1.

FIG. 11. Expression of IL-2 receptor alpha chain CD25 on PBMC followingcyclotide treatment. PBMC were pretreated with cyclosporine A (CsA; 5μg/mL) or different cyclotides (4 μM; T20K, V10A, V10K, T8K) and werecultivated in the presence of media (SC) or were stimulated with PHA-L(10 μg/mL; CTRL). At day 1 (A and B) or day 2 (C and D) aftercultivation, the cells were surface-stained with anti-human CD25 mAbsand were analyzed by flow cytometry. Representative results weredepicted as dot plots (A and C) and results of three independentexperiments are presented as mean and standard deviation (SD) of threeindependent experiments. The asterisks represent significant differencesfrom untreated stimulated cells alone. The percentages indicated in thedot plots represent the CD25⁺ PBMC.

FIG. 12. A. IL-2 secretion from cyclotide-treated activated PBMC. PBMCwere pretreated with cyclosporine A (CsA; 5 μg/mL) or a cyclotide (4 μM;T20K) and were cultivated in the presence of media (SC) or werestimulated with PHA-L (10 μg/mL; CTRL). 24 hours after cultivation, PBMCwere restimulated with PMA/Ionomycin for further 6 hours. Afterwards,the amount of IL-2 was measured in the supernatant by using anELISA-based flow cytometric technique. Data are presented as mean andstandard deviation (SD) of three independent experiments.

B. IL2 release in human T-cells after treatment with cyclotide. HumanT-cells (provided by A. Dohnal, PhD; from CCRI, Vienna) 4×106/mL wereseeded in 96-well flat-bottom plates (100 μL/well) and incubated for twohours at 37° C. before they were stimulated with CsA (5 mg/mL), T20K (4μM) and V10K (4 μM). After another two hours PHA-L (10 μg/mL) was addedto the appropriate wells and incubated over night at 37° C. On the nextday T-cells were re-stimulated with Ionomycin (500 ng/mL) and PMA (50ng/mL) for 6 hours at 37° C. Cells were then centrifuged at 3000 rpm for5 minutes to gain their supernatants. Supernatants of stimulated T-cellswere analyzed for their IL2 release using a human IL-2 ELISA Kit fromeBioscience according to the manufacturer's instructions. The colorreaction was evaluated at an optical density of 450 nm by the microplatereader Synergy H4 (BioTek). PHA-L stimulation of human T-cellsillustrated highest IL2 release, also V10K and PMA+Ionomycin stimulationachieved comparable results, whereas untreated and CsA treated cellsshowed no production of this cytokine. In addition, T-cells incubatedwith the cyclotide T20K demonstrated a significant inhibition of cellproliferation in accordance to the IL2 level.

C. IL-2 gene expression analysis using RT-PCR. Total cellular RNA wasisolated from PHA-L-activated cells that were incubated with medium, CsAor T20K for 4 hours. RT-PCR was carried out using specific primers forindicated gene. The data were normalized to the Ct value of the internalhousekeeping gene 18s rRNA and the relative mRNA level in the untreatedstimulated group was used as calibrator. Data were expressed as mean+SDof three independent experiments.

FIG. 13. Proliferation capacity of cyclotide-treated PBMC in thepresence of exogenous IL-2. CFSE-labelled PBMC were pretreated withcyclosporine A (CsA; 5 μg/mL) or different cyclotides (4 μM; T20K, V10A,V10K, T8K) and were cultivated in the presence of media (SC) or werestimulated with PHA-L (10 μg/mL; CTRL). The cells were cultured withoutexogenous IL-2 (10 U/mL) (A and B) or in the presence of IL-2 (C and D).The CFSE-labelled cells were measured after a 3 day culture period byflow cytometry and representative data are presented in dot plots (A andC). Data are presented as mean and standard deviation (SD) of threeindependent experiments.

FIG. 14. IFN-γ secretion by cyclotide-treated PBMC. Purified PBMC werepreincubated with a cyclotide (4 μM; T20K) or cyclosporine A (CsA; 5μg/mL) and were stimulated with PHA-L (10 μg/mL). Untreated cells wereused as control. Following 24 h or 36 h of cultivation, the cells wererestimulated with PMA/Ionomycin for further 6 hour. The amount of IFN-γwas measured in the supernatant of cultured cells using an ELISA-basedflow cytometric method. The data are presented as mean and standarddeviation (SD) of three independent experiments.

FIG. 15. TNF-alpha secretion from cyclotide-treated PBMC. Purified PBMCwere preincubated with a cyclotide (4 μM; T20K) or cyclosporine A (CsA;5 μg/mL) and were stimulated with PHA-L (10 μg/mL). Untreated cells wereused as control. Following 24 h or 36 h of cultivation, the cells wererestimulated with PMA/Ionomycin for further 6 hour. The amount ofTNF-alpha was measured in the supernatant of cultured cells using anELISA-based flow cytometric method. The data are presented as mean andstandard deviation (SD) of three independent experiments.

FIG. 16. Degranulation capacity of cyclotide-treated activated humanPBMC. PBMC were pretreated with cyclosporine A (CsA; 5 μg/mL) or acyclotide (4 μM; T20K) and were cultivated in the presence of media (SC)or were stimulated with PHA-L (10 μg/mL; CTRL). After 36 hour ofcultivation the cells were restimulated with PMA/Ionomycin for 2.5 hoursin the presence of a CD107a mAbs and GolgiStop reagent to determine theamount of degranulation by flow cytometry. Representative data are shownin dot plots (A) and in (B) data are presented as mean and standarddeviation (SD) of three independent experiments.

FIG. 17. Ca²⁺ release in human Jurkat and T-cells. Jurkat cells (A) andT-cells (B) 1×10⁶ were loaded with 1 μM Fura-2 and 0.02% Puronic F-127for 30 minutes at 37° C. Cells were centrifuged for 5 minutes at 1200rpm and resuspended in media [RPMI 1640 with 10% FCS, penicillin (100U/mL) and streptomycin (100 U/mL)]. 100 μL of cell suspension weretransferred to a black 96-well plate with a clear flat-bottom. Brieflybefore analysis the fluorometer Synergy H4 (BioTek) was tempered to 37°C. The fluorescence time course was then measured with: extinction340/380 nm and emission 510 nm in 30 seconds intervals, continuouslyshaking. Ca²⁺ influx was initiated by adding compounds to the cells(illustrated by the arrow). To receive maximum Ca²⁺ release cells werestimulated with PMA (50 ng/mL) and Ionomycin (500 ng/mL) and T-cellsadditionally with PHA-L (10 μg/mL). For lowest Ca²⁺ levels, cellsremained untreated. CsA (5 mg/mL), T20K (4 μM) and V10K (4 μM)stimulation did not induce a change in Ca²⁺ signaling in Jurkats. Incontrast human primary T-cells demonstrate an increasing Ca²⁺ releaseafter incubation with the cyclotides T20K.

FIG. 18. Immunisation scheme (see also Example 14)

FIG. 19. Effect on clinical EAE score. After induction of EAE, micetreated with T20K and naïve mice were scored every second day, startingat day 10. The naïve group, which received no T20K developed worstdisease course, whereas T20K treated mice showed delayed and minorsymptoms of EAE referred to the time point of cyclotides injection.Especially mice treated seven days before EAE induction demonstratesignificantly the prophylactic effect of the kalata B1 mutant (accordingto Dunnett's multiple comparison test).

FIG. 20. Effect on weight of EAE-induced mice. Weight of immunized micewas measured at day (−7), 0, 7 and on each day besides scoring. Micereceiving cyclotide injections at day (−7) gained weight within the nextdays. Whereas untreated mice or mice which were treated at day 7remained constant or even lost body weight according to the diseasecourse. About day 20 EAE in these two groups ameliorated and thereforethese mice regained body weight.

FIG. 21. Effect on cytokine release of ex vivo isolated PBMC at day 3.Spleenocytes of sacrificed mice were isolated and restimulated withMOG₃₅₋₅₅ (30 μg/mL) for three days or left untreated. Supernatants ofthese cells were used for analyzing cytokine release in ELISAs. In (A)interleukin 2 release was highest in splenic T-cells isolated from naïvemouse group that were restimulated with MOG, correlating with diseasecourse. In T20K (7) treated mice IL2 release was lower than in naïvegroup after MOG stimulation. Spleenocytes from pre-treated mice (T20K−7, 0) show a significant inhibition of the IL2 production (according toDunnett's multiple comparison test). This inhibitory effect could alsobe demonstrated towards the cytokines IL17, IL22 and INFγ in T-cells ofT20K (−7, 0) treated mice, although not significantly (B-D). There washardly any cytokine IL4 detectable (E), opposing a T_(H)2 immuneresponse, which was expected.

FIG. 22. Effect on cytokine release of ex vivo isolated PBMC at day 1and 2. Splenic T-cells of sacrificed naïve mouse group were isolated andstimulated with T20K (4 μM) at different time points, with MOG₃₅₋₅₅ (30μg/mL) and for control purposes with CsA (5 μg/mL) and V10K (4 μM), asindicated here. IL2 release is significantly inhibited after a 48 hincubation of the cells with T20K, independent to the different timepoints of cyclotide addition. Even after 24 h IL2 inhibition isnon-significant to the immune suppressive agent CsA. Also V10K shows ainhibitory capacity towards IL2 release in mouse T-cells after 48 hincubation (A, B). The production of the cytokine IL17 is also inhibitedby T20K after 48 h, related to the time point of compound addition (C,D). Furthermore INFγ and IL22 cytokine release is repressedsignificantly, dependent on the cyclotide addition (E-H). To approvethis EAE T_(H) bias towards T_(H)17 and T_(H)1 cells, IL4 release wasagain analyzed, but this T_(H)2 cytokine was not detectable (I, J), asalready indicated in (D).

FIG. 23. Effect of cyclotides on protein expression of NFAT1c. Human T-cells were incubated with the CsA (5 μg/mL), T20K (4 μM) and V10K (4 μM)for two hours. But instead of stimulating with PHA-L and PMA/ionomycin,one part of the cells was stimulated with PHA-L (10 μg/mL) and the otherwith PMA (50 ng/mL)/ionomycin (500 ng/mL) over night. CsA and T20Kincubation show a reduced signal of NFATc1 compared to the cellsstimulated with V10K, PHA-L and PMA/Ionomycin (A). Splenic T-cellsisolated from naïve mouse group were stimulated as described above.Cells incubated with the cyclotides T20K demonstrate a reduced NFATc1signal compared to cells incubated with V10K and cells stimulated withthe natural antigen MOG.

Although, cells treated with the immunosuppressant compound CsA whichhas NFAc1 as a major molecular target, show a strong signal (B).

FIG. 24. Cellular uptake of T20K. Human T-cells, were incubated with 4μM T20K labeled with FITC to perform fluorescence microscopy. (A)demonstrates an overview of the T-cells with the incorporated cyclotidesT20K in their cytosol. It seems that the peptide is mostly found aroundthe membrane of the nucleus, but also in the membrane of vesicularcompartments, like the Golgi apparatus or the Endoplasmic reticulum (B,C). In contrast incubating Jurkats (D) with the labeled peptide did notshow this intracellular fluorescence, instead the cyclotides stainedonly dead cells.

The Examples illustrate the invention.

EXAMPLE 1: MATERIAL AND METHODS

Extraction Preparation and Purification of Plant Cyclotides.

Oldenlandia affinis (R&S) DC. plants were grown in the glass house atthe Department of Pharmacognosy (University of Vienna) from seeds thatwere obtained as a gift from David Craik (Institute for MolecularBiosciences, University of Queensland). Aerial parts of the plants havebeen harvested and dried. Plant material was pulverized using a rotorgrinder and extracted twice overnight in dichloromethane:methanol (1:1v/v). The extracts were concentrated on a roto-evaporator and werelyophilized. The dried extracts were dissolved in solvent A (ddH₂O with0.1% TFA) and in-batch pre-purified with C₁₈ solid phase extraction(ZEOprep 60 Å, C₁₈ irregular 40-63 μm; ZEOCHEM, Uetikon, Switzerland).To separate the hydrophilic non-cyclotide compounds from the hydrophobiccyclotide compounds, the C₁₈-beads were washed with 10% solvent B (90%acetonitrile in ddH₂O with 0.08% TFA) and eluted with 80% solvent B. Theeluate containing cyclotides was analyzed by MALDI-TOF MS andreconstituted in ddH₂O at 10 mg/mL for biological assays or used fornano LC-MS/MS analysis and further purification. Kalata B1 was purifiedfrom crude O. affinis extract by HPLC using a Perkin Elmer Series 200system with preparative (Phenomenex Jupiter, 10 μm, 300 Å, 250×21.2 mm;8 mL/min) and semi-preparative (Kromasil C₁₈, 5 μm, 100 Å, 250×10 mm; 3mL/min) RP-C₁₈ HPLC columns and linear gradients from 0-80% solvent B in80 min. Eluting peptides were monitored with UV-absorbance (A₂₈₀),collected manually and lyophilized. Purity and quality of kalata B1 wasassessed by analytical HPLC and MALDI-TOF MS.

Nano LC-MS and LC-MS/MS Analysis.

Crude, ZipTip™ prepared or digested plant extracts (C₁₈ pre-purified O.affinis extract, see above) were analyzed by nano LC-MS or LC-MS/MS onan Ultimate 3000 nano HPLC system controlled by Chromeleon 6.8 software(Dionex, Amsterdam, The Netherlands). For LC analysis, samples of O.affinis extract (1-5 μL) were injected, pre-concentrated using DionexPepMap™ C₁₈ cartridges (300 μm×5 mm, 5 μm, 100 Å) and separated bynano-RP-HPLC prior to online MS analysis using a Dionex Acclaim PepMap™C₁₈ column (150 mm×75 μm, 3 μm, 100 Å; 300 nL/min). The mobile phaseconsisted of solvent C (0.1% aqueous formic acid) and solvent D (90/10acetonitrile/0.08% aqueous formic acid). Peptides were eluted using alinear gradient of 4-90% D in 35 min, 5-min hold at 90% D, followed by areturn to 4% D for a 20-min equilibration. For LC-MS/MS analysisaliquots (1-10 μL) of tryptic or endo-GluC digested plant extracts werepre-concentrated and separated by C₁₈ nano LC as described above, usingseveral LC gradients of up to 120 min duration (e.g., 4-60% B in 100min, 60-90% B in 1 min and finally a 5-min hold at 90% B, followed by areturn to 4% B for a 10-min equilibration). Eluated peptides weredirectly introduced into the nanospray source. Mass spectrometryexperiments were performed on a hybrid quadrupole/linear ion trap 4000QTRAP MS/MS system (ABSciex, Foster City, Calif., USA) running with theAnalyst 1.5.1 software package. The 4000 QTRAP equipped with anano-spray source was operated in positive ionization mode. LC-MSanalyses for cyclotide quantification and identification by molecularweight were performed using Enhanced Multiple Scan (EMS) acquisitionwith a scan speed of 1000 amu/sec in the mass range from 400-1400 Da.LC-MS data were analyzed by “LC-MS reconstruct” in the MW range from2700-3500 Da and by using several signal-to-noise filter settings toobtain the molecular weight and validity score of all peptide peaks.LC-MS/MS analyses were performed using Information Dependent Acquisition(IDA). The acquisition protocol used to provide mass spectral data fordatabase searching involved the following procedure: mass profiling ofthe HPLC eluant using EMS; ions over the background threshold weresubjected to examination using the Enhanced Resolution (ER) scan toconfirm charge states of the multiply charged molecular ions. The mostand next most abundant ions in each of these scans with a charge stateof +2 to +4 or with unknown charge were subjected to CID using rollingcollision energy. Enhanced product ion scan was used to collate fragmentions and present the product ion spectrum for subsequent databasesearches.

Enzymatic Digest and Peptide Sequencing Using Database Analysis.

C₁₈ prepurified O. affinis extract cyclotides were prepared for MS/MSsequencing as described earlier (Chen, 2005, J Biol Chem, 280,22395-22405; Ireland, 2006, Biochem J, 400, 1-12). The extract wasreduced, alkylated with iodoacetamide and enzymatic digested usingtrypsin or endo-GluC (Sigma-Aldrich, Austria). Digested peptide extractswere analyzed with nano LC-MS/MS as described above and IDA data wereused for further analysis. Database searching of LC-MS/MS data wascarried out using the ProteinPilot™ software and the Paragon algorithmwith the custom-made ERA database tool for the identification ofcyclotides (Colgrave, 2010, Biopolymers, 94, 592-601).

Relative Quantification of Cyclotides Using Nano LC-MS Analysis.

C₁₈ prepurified O. affinis extract was separated by one dimensional nanoLC-MS as described above. Cyclotide peaks were quantified by relativearea under curve (all peaks at 214 nm absorbance from 15-55 min wereprocessed) using the quantification wizard of Chromeleon 6.8 software.Peaks in the LC chromatogram were identified by molecular weight andretention time from corresponding LC-MS peaks. Quantification wasperformed on five independent LC-MS experiments and relative cyclotideabundance is presented as mean±SEM.

Preparation of Human Peripheral Blood Mononuclear Cells and CellCulture.

Human peripheral blood mononuclear cells (PBMC) were isolated from theblood of healthy adult donors obtained from the Blood Transfusion Centre(University Medical Center, Freiburg, Germany). Venous blood wascentrifuged on a LymphoPrep™ gradient (density: 1.077 g/cm³, 20 min,500×g, 20° C.; Progen, Heidelberg, Germany). Afterwards cells werewashed twice with medium and cell viability and concentration wasdetermined using the trypan blue exclusion test. PBMC were cultured inRPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum(PAA, Coelbe, Germany), 2 mM L-glutamine, 100 U/mL penicillin and 100U/mL streptomycin (all from Invitrogen, Karlsruhe, Germany). The cellswere cultured at 37° C. in a humidified incubator with a 5% CO₂/95% airatmosphere. All experiments conducted on human material were approved bythe Ethics committee of the University of Freiburg.

Alternative Purification of Human Peripheral Mononuclear Cells (PBMCs).

PBMCs were isolated from blood samples of healthy adults that wereprovided by the transfusion center of the university hospital inFreiburg (Germany). Venous blood was diluted 1:2 (v/v) with PBS andcentrifuged with a LymphoPrep-gradient (using 15 ml diluted blood and 20ml LymphoPrep solution); density: 1.077 g/cm³, 20 min, 500×g, 20° C.).The lymphocyte-enriched layer was transferred into a new vessel andwashed three times with PBS and centrifuged again (10 min, twice with300×g and last time with 800 rpm, 20° C.). For the followingexperiments, the cells were either stained with CFSE or diluted withmedium to 4*10⁶ cells/ml. Cells were counted in alight microscope usingtrypan blue staining and a hemocytometer.

Activation and Treatment of PBMCs.

PBMCs (10⁵) were stimulated with anti-human CD3 (clone OKT3) andanti-human CD28 (clone 28.2) mAbs (both from eBioscience, Frankfurt,Germany) for 72 hrs in the presence of medium, the control agentscamptothecin (CPT; 30 μg/mL: Tocris, Eching, Germany) and Triton-X 100(0.5%; Carl Roth, Karlsruhe, Germany) or different concentrations of O.affinis extract, melittin (PolyPeptide, Strasbourg, France) or kalataB1, respectively. After cultivation, the cells were assessed inbioassays as described in the text.

Alternative Activation and Treatment of PBMCs.

Following purification, PBMCs were equilibrated for 2 h at 37° C.Afterwards 100 μl PBMCs (4*10⁶ cells/ml) were pre-incubated in a 96-wellplate for 2 h with CsA (cyclosporine A) or cyclotides, transferred to anew plate and stimulated with 10 μg/ml PHA-L for 1 h. This was followedby washing of each well with 100 μl PBS (centrifugation for 5 min, 1000rpm, 20° C.) and re-suspending the cells in 100 μl medium for furtherassays.

Determination of Cell Proliferation and Cell Division.

For cell proliferation and cell division tracking analysis PBMC wereharvested and washed twice in cold PBS and resuspended in PBS at aconcentration of 5×10⁶ cells/mL. Cells were incubated for 10 min at 37°C. with carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 μM:Sigma-Aldrich, Taufkirchen, Germany). The staining reaction was stoppedby washing twice with complete medium. Afterwards, the cell divisionprogress was analysed using flow cytometric analysis.

Alternative Analysis of Cell Proliferation and Cell Division Using CFSEStaining.

Purified PBMCs (5*10⁶ cells/ml) were incubated with 0.5 mM of thefluorescent dye CFSE (5-carbofluoreszenein-ciacetat-succinylester) for10 min at 37° C. The reaction was stopped using medium, cells werewashed one time with medium by centrifugation (10 min, 300×g, 20° C.)and diluted with medium to 4*10⁶ cells/ml.

Determination of PBMC Apoptosis and Necrosis Using Annexin V andPropidium Iodide Staining.

The levels of apoptosis were determined using the annexin V-FITCapoptosis detection kit (eBioscience, Frankfurt, Germany) according tothe manufacturer's instructions. After annexin V staining, propidiumiodide solution (PI; eBioscience) was added and the cells were incubatedin the dark, followed by a flow cytometric analysis to determine theamount of apoptosis and necrosis. CPT (30 μg/mL) and Triton-X 100 (0.5%)was used as positive controls for apoptosis and necrosis, respectively.

Mice.

C57BL/6 mice (10-16 weeks old) were bred and maintained in the MonashUniversity Animal Services facilities. All experiments were conducted inaccordance with the Australian code of practice for the care and use ofanimals for scientific purposes (NHMRC, 1997), after approval by theMonash University Animal Ethics committee (Clayton/Melbourne,Australia).

Induction and Clinical Assessment of EAE.

A total of 200 μg of the encephalitogenic peptide MOG₃₅₋₅₅(MEVGWYRSPFSRVVHLYRNGK; GL Biochem, Shanghai, China) emulsified in CFA(Sigma) supplemented with 4 mg/ml Mycobacterium tuberculosis (BD) wasinjected subcutaneously into the flanks. Mice were then immediatelyinjected intravenously with 350 ng of pertussis vaccine (List BiologicalLaboratories, Campbell, U.S.A.) and again 48 hr later (Bernard J Mol Med75, 1997, 77-88; Albouz-Abo Eur J Biochem 246, 1997, 59-70; Hvas Scand JImmunol 46, 1997, 195-203; Johns Mol Immunol 34, 1997, 33-38; Menon JNeurochem 69, 1997, 214-222). Animals were monitored daily andneurological impairment was quantified on an arbitrary clinical scale:0, no detectable impairment; 1, flaccid tail; 2, hind limb weakness; 3,hind limb paralysis; 4, hind limb paralysis and ascending paralysis; 5,moribund or deceased (Liu Nat Med 4, 78-83 1998; Slavin Autoimmunity 28,109-120 1998). Under recommendation of the animal ethics committee, micewere euthanised after reaching a clinical score of 4.

Antibodies and Recombinant Proteins.

The mouse anti-MOG mAb (clone 8-18C5) was purified from hybridomaculture supernatants on Protein G-Sepharose 4 Fast Flow column (GEHealthcare) according to the manufacturer's instructions. Antiserum toMOG₃₅₋₅₅ peptide (Ichikawa Int Immunol 8, 1996, 1667-1674; Ichikawa JImmunol 157, 1996, 919-926) was raised in rabbits by procedures similarto those described previously (Bernard Clin Exp Immunol 52, 1983,98-106; Pedersen J Neuroimmunol 5, 1983, 251-259). The extracellulardomain of mouse MOG (amino acid residues 1-117 of the mature protein)(rMOG) was produced in the E. coli strain M15pREP4 using the pQE9expression vector (Qiagen, Australia) to incorporate an amino-terminalhistidine tag as per manufacturer's instructions. A clarified bacteriallysate containing rMOG was loaded onto a Ni-NTA Superflow (Qiagen,Australia) column under denaturing conditions (6 M Guanidine-HCl, 100 mMNaH₂PO₄, 10 mM Tris pH 8.0,) as per the manufacturer's instructionsusing a BioLogic LP Chromatography System (Bio-Rad Laboratories,Australia). Bound protein was washed sequentially with Buffer A (8M Urea100 mM NaH₂PO₄, 10 mM Tris pH 8.0), Buffer A (at pH6.3), 10 mM Tris pH8/60% iso-propanol (to remove endotoxin) and again with Buffer A.Refolding of the bound protein was carried out by applying a lineargradient of Buffer A containing 14 mM 2-mercaptoethanol (100%-0%) vs.Buffer B (100 mM NaH₂PO₄, 10 mM Tris pH 8.0, 2 mM reduced glutathione,0.2 mM oxidised glutathione) (0%-100%). This was followed by a secondlinear gradient of Buffer B (100%-0%) vs. Buffer C (100 mM NaH₂PO₄, 10mM Tris pH 8.0) (0%-100%). The bound protein was eluted using Buffer Ccontaining 300 mM Imidazole, then extensively dialysed against 50 mMNaCl/10 mM Tris pH 8. Protein concentration and purity were estimatedusing a Micro BCA assay (Bio-Rad Laboratories, Australia) and SDS-PAGE,respectively. The protein produced was varified as rMOG by western blotanalysis using antibodies specific for native MOG. Endotoxin levels weredetermined using a Limulus Amebocyte Lysate assay (Associates of CapeCod, Falmouth, Mass.).

Vaccination with MOG Peptide.

200 μg of the MOG peptide were emulsified with an equal volume of IFA(Difco) and injected subcutaneously in the upper flanks (100 μl dividedequally) three weeks prior to the encephalitogenic challenge. This wasfollowed by two more injections at weekly intervals (200 μg/IFA/100 μl).

Histopathology and Assessment of Inflammation, Demyelination and AxonalDamage.

At the completion of the experiments, mice were anesthetized, theirblood collected (for subsequent antibody determination) and brain andspinal cord carefully removed, prior to immersion in a 4%paraformaldehyde, 0.1 M phosphate buffer solution. Segments of brain,cerebellum and spinal cord were embedded in paraffin. Sections werestained with haemotoxylin-eosin, Luxol fast blue and Bielshowsky forevidence of inflammation, demyelination and axonal damage, respectively(McQualter 2001 J Exp Med. October 1; 194(7), 873-82). Semiquantitativehistological evaluation for inflammation and demyelination was performedand scored in a blind fashion as follows: 0, no inflammation; 1,cellular infiltrate only in the perivascular areas and meninges; 2, mildcellular infiltrate in parenchyma; 3, moderate cellular infiltrate inparenchyma; and 4, severe cellular infiltrate in parenchyma (BettadapuraJ Neurochem 70, 199, 1593-1599 8; Okuda J Neuroimmunol 131, 2002,115-125).

MOG-Specific Antibody Determination.

Antibody activity to rMOG and MOG₃₅₋₅₅ in mouse sera was measured byELISA, as previously described Ichikawa Cell Immunol 191, 1999, 97-104).Briefly, serum was collected at the end of the experiments and tested byELISA with rMOG and MOG₃₅₋₅₅ peptide-coated plates (Maxisorp, Nunc).

T Cell Proliferation and Cytokine Production.

Spleens were taken from mice sacrificed 32-46 days after MOG₃₅₋₅₅immunization. Cells were gently dispersed through a 70 μm nylon mesh(BD) into a single cell suspension, washed and cultured at 2.5×10⁶cells/ml in complete RPMI (RPMI 1640 containing 10% heat-inactivatedfetal calf serum (Sigma), 2 mM L-glutamine, 100 U/ml of penicillin, 100μg/ml of streptomycin, 50 μm 2-mercaptoethanol and 1 mm sodium pyruvate.Two hundred microliters of cell suspensions were then added to 96 wellmicrotitre plates either alone, with MOG₃₅₋₅₅ (20 μg/ml) or anti-CD3εand anti-CD 28 (20 μg/ml each) and incubated for 66 h at 37° C. with 5%CO₂. Ten microliters of [³H]thymidine (1 μCi/well; Amersham, Australia;diluted 1/10 in media) were added to each well for the last 18 h. Plateswere harvested onto glass fibre filters and a drop of MicroscintScintillant (Perkin Elmer) was added to each well. Counts were readusing a Top Count NXT Scintillation Counter (Perkin Elmer). Presentedvalues are the mean of three wells. For cytokine assays, 2 ml of cells(5×10⁶ cells/ml) from spleens isolated 32-46 days after immunizationwere added to 24 well plates either alone or with MOG₃₅₋₅₅ (10 μg/ml) orwith anti-CD3ε and anti-CD 28 (20 μg/ml each). Supernatants werecollected at 48 and 72 h. Quantitation of mouse cytokine contentincorporating Th1, Th2 cytokines and chemokines (IFNγ, IL-2, IL-3, IL-4,IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, GM-CSF, KC, MCP-1, MIG, andTNF) were simultaneously determined using a multiplexed bead assay(Cytometric Bead Array Flex sets [CBA]) according to the manufacturer'srecommended protocol (Becton Dickinson). Acquisition of 4500 events wasperformed using a FACScanto II flow cytometer (Becton Dickinson, SanJose, USA) and Diva software and data analysed and fitted to a4-parameter logistic equation using the FCAP array software (Soft Flow,Pécs, Hungary). Minimum detection levels of each cytokine were: IFNγ,5.2 μg/ml; IL-2, 1.5 μg/ml; IL-3, 4.2 μg/ml; IL-4, 0.8 μg/ml; IL-5, 4.8μg/ml; IL-6, 6.5 μg/ml; IL-9, 10.5 μg/ml; IL-10, 16.4 μg/ml; IL-12p70,9.2 μg/ml; IL-13, 7.3 μg/ml; GM-CSF, 9.9 μg/ml; KC, 16.2 μg/ml; MCP-1,29 μg/ml; MIG, 11.4 μg/ml and TNF, 17.1 μg/ml.

IL-2 Surface Receptor Analysis.

Activated cells were transferred into a 96-well plate, centrifuged (5min, 1000 rpm, 20° C.), washed one time with 100 μl FACS-buffer andstained with CD25 PE for 15 min at 4° C. Then cells were washed twicewith FCS-buffer and resuspended in 100 μl FACS-buffer, transferred intoFACS vials with a total volume of 250 μl and the expression of IL2surface receptor CD25 was measured by FACS analysis using a FACSCaliburinstrument (BD Biosciences).

Determination of Cytokine Release Using ELISA.

Activated cells were resuspended in 50 μl of medium, transferred into a96-well plate and treated with 10 μg/ml PHA-L. After incubation for 24 hcells were re-stimulated with PMA (50 ng/ml) und ionomycin (500 ng/ml)for 6 h. Next, the cells were transferred into Eppendorf tubes,centrifuged (5 min, 3000 RPM, 20° C.) und 50 μl of the supernatant wasagain transferred into new tubes and stored at −20° C. Production ofcytokines was measured and quantified using the FlowCytomix™ kitaccording to manufacturer's instructions.

CD107a—Degranulation Analysis.

Activated cells were grown for 36 h and then treated for re-stimulationwith PMA (50 ng/ml) und ionomycin (500 ng/ml) and stained with CD107aPE. After 1 h the reaction was stopped with 2 μl Golgi-Stop (1:10) andincubated for 2.5 h at 37° C. The cells were transferred into a 96-wellplate, centrifuged (5 min, 1000 RPM, 20° C.) and washed with 100 μlFACS-buffer. Afterwards, PBMCs were stained with CD8 PE-Cy5 for 15 minat 4° C. and following to wash cycles with FACS-buffer the cells wereresuspended in 100 μl, transferred into FACS vials with a total volumeof 250 μl and the degranulation was measured by FACS analysis.

Intracellular Production of IFN-Gamma and TNF-Alpha.

Activated cells were grown for 36 h and then treated for re-stimulationwith PMA (50 ng/ml), ionomycin (500 ng/ml) and brefeldin A for 6 h at37° C. After transferring the cells into a 96-well plate, they werecentrifuged (5 min, 1000 RPM, 20° C.) and washed with 100 μlFACS-buffer. Afterwards, PBMCs were stained with CD8 PE-Cy5 for 15 minat 4° C. and washed again twice with FACS-buffer. The cells were treatedwith 50 μl of 4% paraformaldehyde for 10 min at 4° C., washed twice with100 μl FACS-buffer and then permeabilized by incubation with 100 μlPerm/Wash solution (1:10) for 15 min at 4° C. After centrifugation (5min, 1000 rpm, 20° C.), PBMCs were incubated with IFN-gamma PE orTNF-alpha PE, respectively, for 30 min at 4° C. Free antibodies werewashed away with Perm/Wash and PBMCs were re-suspended in 100 μlFACS-buffer. Production of IFN-gamma and TNF-alpha was individuallydetermined by FACS analysis.

Total RNA Extraction and Reverse Transcription.

Total RNA was extracted from controls or treated cells (2×10⁶) frozen at−80° C. RNA-purification was performed according to the manufacturer'sinstructions for the RNeasy mini and Rnase-Free Dnase Set digestion kits(Qiagen, Hilden, Germany). The quantity and purity of extracted RNA wasmeasured by spectrophotometry (Nanodrop, Peqlab, Erlangen, Germany) andpurified RNA was reverse transcribed using the RT² First Stand Kit(Qiagen, Hilden, Germany).

Real-Time PCR.

RT-PCR reactions were carried out on a BioRad MyiQ (BioRad, Munich,Germany) in a final volume of 25 μL using RT² qPCR Primer Assay (forIL-2) and SYBR® Green qPCR Mastermix (both from Qiagen, Hilden,Germany). Each determination was done in duplicate and the housekeepinggene 18s rRNA was used as an internal control. The real-time thermalcycler program consisted of an initial denaturation step at 95° C. for10 min followed by a two-step cycling program with 40 cycles (95° C., 15s; and 60° C., 60 s). Results were expressed as relative gene expressionof IL-2 and were determined by comparative Ct method. The data werenormalized to the Ct value of the internal housekeeping gene 18s rRNAand the relative mRNA level in the untreated group (untreatedPHA-L-activated) was used as calibrator.

Data Analysis and Statistical Analysis.

For FIG. 10, statistical analysis were performed using the Student's ttest, with P values <0.05 considered significant. All other graphs wereprepared using GraphPad Prism™ software and data are presented asmean±standard error (SEM). Where applicable, data were statisticallyanalyzed using one-way ANOVA Kruskal Wallis test and Dunn's multiplecomparison post analysis.

FACS graphs and results were prepared using CellQuest Pro Software (BDBiosciences) and are presented as mean+STDEV or SEM. All data pertainingexamples 8-12 were statistically evaluated by ANOVA and Dunnet's posthoc-test using SPSS v19.0 (IBM, NY, USA).

EXAMPLE 2: CHEMICAL ANALYSIS OF OLDENLANDIA AFFINIS PLANT EXTRACT

The crude extract of the coffee-family plant Oldenlandia affinis waschemically analysed using a rapid peptidomics workflow utilisingnano-LC-MS, peptide reconstruct with database identification and MS/MSautomated sequence analysis to determine its cyclotide content.

O. affinis plants were grown and the aerial parts were isolatedaccording to well-known laboratory protocols using overnight extractionwith dichloromethane and methanol followed by C₁₈ solid phase extractionof the aqueous part. This standard procedure commonly yields many gramsof crude cyclotide-extract per kilogram of fresh plant leaf weight(Gruber, 2007, Toxicon, 49, 561-575; Gran, 1970, Medd Nor Farm Selsk,12, 173-180), while the content of various cyclotides depends on thegrowth conditions (e.g., habitat) of the plants and other environmentalfactors (Trabi, 2004, J Nat Prod, 67, 806-810; Seydel, 2007, Appl.Microb. Biotechnol., 77, 275-284).

Generally, amino acid sequencing is only feasible from pure orsemi-purified cyclotide fractions. Therefore, an alternative peptidomicsapproach was used to dissect the cyclotide content from a crude plantextract by combining nanoflow LC-MS and peptide reconstruction(identification by molecular weight) as well as proteolytic digestion,LC-MS/MS and automated database analysis (identification by amino acidsequence) using the recently reported ERA cyclotide database tool(Colgrave, 2010, Biopolymers, 94, 592-601). The crude cyclotide extractwas analyzed with various linear gradients on reversed-phase C₁₈ nano LCcoupled online to an electrospray ionization hybridtriple-quadrupole/linear ion-trap (ESI-QqLIT) mass spectrometer, whichwas operated in enhanced MS mode with scan speeds of 1000 and 4000amu/sec, respectively. Application of an automated LC-MS reconstructtool yielded initially a few hundred of peptide masses in the range from2700-3500 Da (typical MW for cyclotides). The high number likelyaccounts for some false-positive hits due to the inclusion of lowabundant data in the calculation. Hence, the signal-to-noise factor inthe algorithm was adjusted and usually between 50-100 reconstructedpeptide masses with significant scores above 0.99 were obtained.Representative LC-MS reconstructed data (of at least three independentexperiments) are listed in Table 4. A total of 72 peptide masses in therange from 2700-3500 Da were identified. By comparing those peptidemasses to the database of cyclotides (CyBase (Wang, 2008, Nucleic AcidsRes, 36, D206-210)), 23 known O. affinis cyclotides, 24 peptide massesthat correspond to peptides from other cyclotide plant species and 25new (not previously described) cyclotide masses were identified. LC-MSexperiments were further analyzed with manual peptide reconstruction byextracting the doubly- and triply-charged ions of respective cyclotidepeaks and by calculation of the average molecular weight (unpublisheddata). The manual analysis was useful as an internal control to ensurethe integrity of the generated automated data.

In addition to the analysis of O. affinis cyclotides by molecular weightand database comparison, a number of chemical modifications of the crudeextract, i.e. reduction and alkylation followed by trypsin and endo-GluCproteolysis, were performed. Due to the structural nature and highstability of cyclotides these chemical modifications are necessary toyield amenable precursor ions for MS/MS sequencing. The modified anddigested mixtures were analyzed with a peptidomics workflow utilizingnano LC and peptide sequencing by Information Dependent Acquisition (forfurther details see the Methods Section). The resulting MS and MS/MSdata were used for automated cyclotide identification using theParagonTM algorithm with a custom-made ERA cyclotide database (a toolthat is freely available on the web). Using this cyclotide peptidomicsanalysis, 14 known cyclotides could be identified by amino acid sequence(see Table 5). In summary, using the above described peptidomicsworkflow nearly all currently known cyclotides and an even greaternumber of novel peptide masses corresponding to other known or novelcyclotides (by molecular weight) could be identified in crude cyclotideextract from the plant O. affinis (see Table 1).

The combination of nano LC-MS/MS and LC-MS reconstruction, as well asautomated database searching is a rapid and useful technique for theidentification of cyclotides in crude extracts. Compared to an earlierstudy from Plan et al. (Plan, 2007, ChemBioChem, 8, 1001-1011), whichdescribed the first cyclotide fingerprint of O. affinis using classicalpeptide purification via analytical HPLC and offline MS/MS sequencing, 8additional known cyclotides have been identified and a list of ˜50peptide masses has been provided corresponding to cyclotides of whichsome can be identified by peptide fingerprint analysis in CyBase (thecyclotide database (Wang, 2008, Nucleic Acids Res, 36, D206-210)). Thissuggests that the number of cyclotides to be found in a single speciesmay be >70 and is, therefore, at least twice the number than earlieranticipated (on average 34 cyclotides per species (Gruber, 2008, PlantCell, 20, 2471-2483). This, of course, has a huge impact on thedetermination of the overall number of cyclotides in the plant kingdomand consequently would lead to a necessary revision of the number ofnovel cyclotides to be discovered in plants.

EXAMPLE 3: ANTI-PROLIFERATIVE EFFECTS OF O. AFFINIS CYCLOTIDE EXTRACT

After completion of the chemical analysis, different concentrations ofthe crude O. affinis cyclotide extract were tested for itsanti-proliferative capacity on activated human primary PBMC (FIG. 2). Byusing flow cytometric-based forward-side-scatter analysis, it wasdemonstrated that the extract exhibits a dose-dependent (50-100 μg/mL)decrease of activated proliferating PBMC compared to untreatedstimulated control (FIGS. 2A and B). Simultaneously, a constant contentof viable, resting PBMC, without accumulation of dead cells wereobserved, showing that the applied concentrations of the cyclotideextract are not harmful to the cells. Above this concentration range,the extract showed an increasing cytotoxic effect. Along this line,camptothecin (CPT, 30 μg/mL), which was used as positive inhibitoryproliferation control, induced a high proportion of dead cells,indicating that the observed anti-proliferative effect, in contrast tothe O. affinis cyclotide extract, was mainly due to cytotoxicity.

The impact of the crude O. affinis cyclotide preparation on the celldivision level of activated PBMC was further evaluated. For thispurpose, the cells were labeled with the dye carboxyfluoresceindiacetate succinimidyl ester (CFSE), which does not influence theviability of the stained cells and is inherited by daughter cells aftercell division and each dividing cell consequently loses fluorescentintensity. These data, shown in FIGS. 2C and D, indicate that theextract caused a dose-dependent inhibition of cell division of activatedPBMC, which confirms that the crude O. affinis cyclotide preparation hasthe ability to inhibit PBMC proliferation without cell damage.

EXAMPLE 4: RELATIVE QUANTIFICATION OF CYCLOTIDES AND ISOLATION OF KALATAB1

Since promising anti-proliferative activity of the total cyclotideextract from O. affinis was obtained, the relative amount of the majorcyclotides was determined and the main components for biologicalcharacterization were further purified. For this purpose the crudecyclotide extract was used for quantitative nano LC-MS analysis, similaras described above. Diluted aliquots of the extract were separated bynano C₁₈ RP-HPLC coupled online to the mass spectrometer. Elutedpeptides were monitored both with absorbance at 214 nm and by molecularweight. The area-under-curve of the major cyclotide peaks in O. affiniswas determined by automated integration (and if necessary manualpost-processing). The relative quantification analysis of the cyclotidecontent has been carried out from five independent LC-MS experiments(see Table 6) and a representative O. affinis elution profile,indicating the major cyclotide peaks and their relative abundance(mean±SEM), is shown in FIG. 3.

As presented above, and in agreement with earlier studies (Plan, 2007,ChemBioChem, 8, 1001-1011), the cyclotides kalata B1 and kalata B2 arethe main peptide components, accounting for approx. 34% of the overallcyclotide content in O. affinis. Kalata B1 and B2 differ by only fiveamino acid positions (see FIG. 6), namely Val to Phe (loop 2) andconservative replacements of Thr to Ser (loop 4), Ser to Thr (loop 5),Val to lle (in loop 5) and Asn to Asp (in loop 6) in kalata B2. Sincethese substitutions have no significant structural consequences(RMSD_(backbone kB1/kB2)=0.599 Å, see FIG. 6) and since the two peptideshave a similar bioactivity profile (Gruber, 2007, Toxicon, 49, 561-575),kalata B1 (comprising ˜14% of total extract) was used for furtherbiological analysis and its anti-proliferative potential on activatedhuman primary PBMC.

EXAMPLE 5: ANTI-PROLIFERATIVE AND CYTOTOXIC EFFECTS OF KALATA B1

To analyze whether kalata B1 has the capacity to inhibit theproliferation of activated human primary PBMC, the cells were labeledwith the fluorescent dye CFSE and analyzed the cell division propertiesin the presence of the kalata B1 concentrations in the range from 1.8 to14 μM using flow cytometry. After exposure of PBMC to kalata B1, adose-dependent decrease of the cell division capacity was observed, ascompared to untreated stimulated PBMC controls, as shown in FIG. 4. Theinhibitory concentration IC₅₀ for the anti-proliferative effect ofkalata B1 was 3.9±0.5 μM (FIG. 7), which compares to other effects ofkalata B1, such as nematocidal (Huang, 2010, J Biol Chem, 285,10797-10805) and cytotoxic activities (Svangard, 2004, J Nat Prod, 67,144-147; Lindholm, 2002, Mol Cancer Ther, 1, 365-369; Daly, 2004, FEBSLett, 574, 69-72) as has been summarized in Table 3.

To analyze whether the anti-proliferative effect was due to celldamaging, the influence of kalata B1 on the induction of PBMC apoptosisor necrosis was examined (FIG. 5). Cellular apoptotic and necrotichallmarks were measured by using inter-nucleosomal DNA fragmentation(subG1⁺ cells) assay and phosphatidylserine surface analysis through asingle and combinatory annexin V and propidium iodide staining. Thisdouble staining process allowed the discrimination between viable(annexin⁻/PI⁻), apoptotic (annexin⁺/PI⁻ and annexin⁺/PI⁺) or necrotic(annexin⁻/PI⁺) cells. The data shown in FIG. 5A to C demonstrate thatkalata B1 had no significant influence on the induction of apoptosis.Necrosis was slightly increased at higher concentration (14 μM) ofkalata B1, compared to untreated control (FIG. 5D). The positivecontrols for apoptosis and necrosis, CPT (30 μg/mL) and detergent(Triton-X 100), respectively, significantly increased the fractions ofthese cells.

The anti-proliferative activity of kalata B1 triggered validation andcontrol experiments to determine the nature of the observed effect.Cytometric-based forward-side-scatter analysis (data not shown) providedsolid evidence that the anti-proliferative effect induced by thecyclotide does not cause cell death by either apoptosis or necrosis, butinhibits the growth of the lymphocytes in a cytostatic fashion.Concentrations higher than 14 μM of the peptide are cytotoxic to thecells (data not shown). This was expected since kalata B1 has earlierbeen reported to cause hemolysis and membrane disruption atconcentrations above ˜50 μM (Barry, 2003, Biochemistry, 42, 6688-6695;Henriques, 2011, J Biol Chem, 286, 24231-24241). Therefore, controlexperiments were performed with the honeybee venom component melittin, acommonly used strong membrane disrupting peptide agent.

Concentrations were tested, at which cytotoxic effects on humanlymphocytes were described in the literature, to ensure that ourexperimental setup was sensitive enough to detect possible cytotoxiceffects of kalata B1 (Pratt, 2005, In Vitro Cell Dev Biol Anim, 41,349-355) (see FIG. 8). The data revealed that in contrast to kalata B1,melittin induced a decrease of proliferating PBMC at 1.6 μM (FIG. 8A),but this effect was mainly due to the induction of apoptosis, asindicated by the results of the inter-nucleosomal DNA fragmentationanalysis (FIG. 8B) and by induction of specific apoptotic cells at theseconcentrations (FIG. 8C). In addition, there was a slight effect onnecrosis induction at high concentrations of melittin (FIG. 8D).

From these control data, it was concluded that kalata B1, in contrast tomelittin, has an anti-proliferative capacity, which is not due tocytotoxic effects and the membrane lysing capacity of kalata B1, asotherwise one would have expected similar observations from the muchmore potent cytotoxic peptide melittin. The proof of anti-proliferativeeffects by holding the cells in an “inactive” state at which they arestill viable, but aren't able to grow and proliferate without causingcell death in a certain dose range is a crucial precondition to classifya substance as immunosuppressant, because cytotoxicity would cause sideeffects.

EXAMPLE 6: TEST OF CYCLOTIDE MUTANTS/VARIANTS IN ANTI-PROLIFERATIVEASSAYS ON PBMCS AND ISOLATED T-LYMPHOCYTES

The anti-proliferative effect of cyclotide mutants/variants was testedaccording to Example 5. In brief, CFSE-labelled PBMCs, ormagnetic-purified CD3+ lymphocytes were stimulated with anti-CD3/28mAbs, in the presence of medium (ctrl), camptothecin (CPT, 30 μg/mL),cyclosporin A (CsA, 1 μg/mL) or different concentrations of cyclotides(1.8-14 μM) for 72 h. Afterwards the cell proliferation was assessed byanalysing cell division using flow cytometric-based histogram analysis.The following peptides (1.8-14 μM) on both PBMCs and CD3-purifiedT-lymphocytes (n≥2) have been tested:

Kalata B1: GLPVCGETCVGGTCNTPGCTCSWPVCTRN Kalata B2:GLPVCGETCFGGTCNTPGCSCTWPICTRD D-kalataB2: all-DGLPVCGETCFGGTCNTPGCSCTWPICTRD Kalata T8K: GLPVCGEKCVGGTCNTPGCTCSWPVCTRNKalata V10A: GLPVCGETCAGGTCNTPGCTCSWPVCTRN Kalata V10K:GLPVCGETCKGGTCNTPGCTCSWPVCTRN Kalata G18K: GLPVCGETCVGGTCNTPKCTCSWPVCTRNKalata N29K: GLPVCGETCVGGTCNTPGCTCSWPVCTRK Kalata T20K, G1K:KLPVCGETCVGGTCNTPGCKCSWPVCTRN Kalata T20K: GLPVCGETCVGGTCNTPGCKCSWPVCTRN

The corresponding IC₅₀ values can be found in Table 2:

TABLE 2 Comparison of kalata B1 (and other cyclotides) inhibitoryeffects on PBMC and CD3 purified T-lymphocyte proliferation. Relativeactivity in other assays IC₅₀ (μM) ± (fold difference to kB1) PeptideSTDEV nematocidal hemolytic insecticidal PBMCs Kalata B1 2.9 ± 1.3^(a)1.0 0.7 1.0 Kalata B2 0.2 ± 0.1^(c) — — — all-D kalata 2.3 ± 0.8^(c) — —— B2 Kalata B1 not active (n.a.)^(b) <0.2  T8A: 0.1 0.2 T8K V10An.a.^(b) — 0.5 1.1 V10K n.a.^(b) <0.2  — — G18K 4.4 ± 0.5^(b) 2.4 G18A:0.6 1.2 N29K 3.2 ± 0.6^(b) 7.0/3.8 N29A: 0.5 1.0 T20K, G1K 1.9 ±0.1*^(b) 6.5/6.8 — — (cytotoxic) T20K 1.9 ± 0.6^(c) 3.0/2.6 — — MCo59n.a.^(b) — — — MCo-CC1 n.a.^(b) — — — MCo-CC2 n.a.^(b) — — — CD3purified lymphocytes Kalata B1 2.4 ± 0.5^(d) — — — Kalata B2 0.6 ±0.02^(d) — — — all-D kalata 2.9 ± 0.4^(d) — — — B2 G18K 3.2 ± 1.8^(c) —— — N29K 2.1 ± 0.9^(c) — — — T20K, G1K 1.1 ± 0.7^(c) — — — (cytotoxic)T20K 2.7 ± 0.6^(d) — — — *this compound is cytotoxic at 14 μM; all datahave been normalized and analyzed with non-linear regression (fixedslope) using Graph Pad, ^(a)n = 7, ^(b)n = 4, ^(c)n = 3, ^(d)n = 2;peptides other than kalata B1, have been supplied by David Craik(Institute for Molecular Bioscience, Australia).

EXAMPLE 7: IN VIVO ACTIVITY IN EAE MOUSE MODEL OF MS

The in vivo activity of cyclotides in the EAE mouse model of MS weretested, as described previously (Okuda J Interferon Cytokine Res 18,1998, 415-421). The ability of mice to recover from motor deficit afterdeveloping a chronic progressive form of EAE was examined by vaccinatingthe mice with kalata B1. MOG MS-like disease model in C57BL/6 mice(Bernard J Mol Med 75, 1997, 77-88) was used, where adult female C57BL/6(10-12 weeks old) mice were vaccinated with three successivesubcutaneous (sc) injections of cyclotides (200 mg each time) inincomplete Freund's adjuvant (IFA) at weekly intervals before EAE wasinduced with MOG₃₅₋₅₅. Control mice were similarly treated but receivedPBS in IFA. Animals were assessed daily for clinical signs of EAE for aperiod of 43 days.

Vaccination with kalata B1 resulted in a reduction in the incidence andseverity of EAE (FIG. 10A). Mice treated with kalata B1, displayedsignificantly milder clinical signs (mean cumulative score 42.2±13.0;p<0.01) as compared to the PBS control group (cumulative score:96.6±7.1; disease duration: 29.1±0.9).

The influence of kalata B1 vaccination on the formation of CNSinflammatory and demyelinating lesions was examined by histologicalstudies of fixed tissue using haemotoxylin/eosin, Luxol fast blue (LFB)and Bielshowsky silver staining. The CNS of all mice treated with PBSshowed extensive inflammatory lesions, characterized by mononuclearinflammatory cells, which were particularly florid in the cerebellum andspinal cord (FIG. 10B). LFB and Bielshowsky silver staining revealedmarked myelin loss and severe axonal injury, respectively, particularlyaround the lesioned tissue in all three CNS regions examined. Kalata B1treated mice displayed some improvement in disease severity as judged bydecrease in histological lesions of EAE (FIG. 10B).

The capacity of spleen cells to proliferate in response to theencephalitogen MOG₃₅₋₅₅ to determine whether the suppressive effect onEAE following vaccination with kalata B1 was associated with a decreasein MOG-specific T cell responses. Furthermore, to address whether thissuppression of EAE was antigen specific and/or the result of a defect inthe activation or function of T-cells, the same population ofsplenocytes was stimulated by the polyclonal activators, anti-CD3 andanti-CD28 antibodies. FIG. 10C shows that regardless of the treatmentregimen, splenocytes from all vaccinated mice proliferated to MOG withstimulation indices (SI) of 2.9±0.4 and 2.7±0.5 for groups treated withkalata B1 and PBS, respectively. These splenocytes displayed strongproliferative responses to the anti-CD3/CD28 antibodies with SI rangingfrom 17 to 47.

Whether the suppression of EAE in mice vaccinated with kalata B1 wasassociated with a decrease in the production of specific antibodies toMOG was examined. Accordingly, sera from kalata B1 and PBS treated micewere collected at the completion of the experiment (Day 43) and testedfor their reactivity to MOG₃₅₋₅₅. As indicated in FIG. 10D, anti-MOGantibodies were detected in all sera regardless of the vaccinationregimen.

It is well established that the development of EAE is associated withthe secretion of proinflammatory cytokines by CNS-antigen specific Tcells (Owens Curr Opin Neurol 16, 2003, 259-265). Since the suppressionof EAE following kalata B1 vaccination was not associated with adecrease in T cell reactivity to MOG, it was investigated whetherMOG-reactive T cells in protected animals may have switched to ananti-inflammatory T cell phenotype. Accordingly, conditioned mediagenerated from in vitro stimulated and non-stimulated spleen cellcultures were assessed in cytokine bead array assays. A total of 15cytokines were analysed simultaneously, including, IL2, IL3, IL4, IL5,IL6, IL9, IL10, IL12p70, IL13, IFNγ, GM-CSF, KC, MCP1, MIG and TNFα.There were no marked changes in cytokine content in MOG₃₅₋₅₅ orCD3/38-stimulated supernatants between cyclotide and control animalgroups (data not shown). In contrast, significantly reduced levels ofthe chemokine MIG known to play a role in T cell trafficking and TNFα, apro-inflammatory cytokine known to be involved in the pathogenesis ofEAE Nicholson Curr Opin Immunol 8, 1996, 837-842) were demonstrated innon-stimulated spleen cell supernatants generated from animals treatedwith kalata B1 (FIGS. 10E and 10F). On the basis of this cytokineprofile, it can be deduced that vaccination with cyclotide, leads to theproduction of an anti-inflammatory T response.

EXAMPLE 8: INFLUENCE/EFFECT OF VARIOUS CYCLOTIDES ON THE EXPRESSION OFIL-2-ALPHA-CHAIN CD25

Amongst other pathways, T-cell proliferation is determined by binding ofthe cytokine IL-2 to its cell surface receptor. Therefore the influenceof cyclotides on the expression of the IL-2 receptor was tested. Thetest compounds were T20K, V10A, V10K and T8K and hence PBMCs weretreated with these cyclotides, following stimulation with PHA-L in orderto determine the expression of the IL-2 surface receptors CD25 after 24and 48 hours of cultivation, respectively, using FACS analysis (FIG.11). As control substance CsA was used. Treatment of PBMCs with CsAleads to a reduction in CD25 surface expression and yields 76%±10.7after 24 hours and treatment with T20K yields 79%±10.1 as compared tountreated cells, i.e. stimulated PBMCs (CTRL, 100%) (FIG. 11B).Treatment with V10A yields 114%±12.5, V10K yields 112%±16.3 and T8Kyields 114%±17.3 CD25 surface expression after 24 h as compared to thecontrol (FIG. 11B). This trend continues after 48 h, i.e. the CD25expression is further reduced by treatment with CsA (62%±7.3) and T20K(46%±18.2) whereas treatment with V10A, V10K und T8K leads to nosignificant change in receptorexpression (FIG. 11C und D). In summary,treatment with CsA (p≤0.01) and the cyclotide T20K (p≤0.001) leads to asignificant reduction of CD25 expression, whereas the cyclotides V10A,V10K und T8K do not influence the expression level of the CD25 receptor.

EXAMPLE 9: INFLUENCE OF CYCLOTIDES ON IL-2 RELEASE AND GENE EXPRESSION

To analyze the mechanism of cyclotide-mediated anti-proliferation ofT-lymphocytes, their effect on the direct release of IL-2 in PBMCs wasdetermined. The cells were treated with a cyclotide and activated withPHA-L. After 24 h the cells were re-stimulated with PMA and ionomycinand the IL-2 concentration in the supernatant (released IL-2) wasmeasured with an ELISA-based FACS methodology (FIG. 12A). The IL-2release was significantly (p≤0.01) reduced by treatment with CsA(18%±15.7) and T20K (24%±18.6) as compared to the control cells. Thecyclotide V10K had no effect on the release of IL-2 (data not shown).

Moreover, supernatants of stimulated T-cells were analyzed for their IL2release using a human IL-2 ELISA Kit from eBioscience according to themanufacturer's instructions. The color reaction was evaluated at anoptical density of 450 nm by the microplate reader Synergy H4 (BioTek)(FIG. 12B).

To determine whether cyclotides have an impact at the gene expressionlevel of the, il-2 gene expression (as control we used 18s rRNA) in PBMCcells was investigated by quantitative real-time PCR (FIG. 12C).Cyclotide T20K clearly decreases the level of IL-2 mRNA in contrast tothe control, whereby as positive inhibition control we used cyclosporineA.

EXAMPLE 10: INFLUENCE OF EXOGENOUS IL-2 ADDITION TO CYCLOTIDE-TREATEDPBMCS

To determine the validity of the significant reduction of IL-2 releaseafter cyclotide treatment, the influence of exogenous addition of IL-2post treatment was tested. If IL-2 synthesis is reduced by treatmentwith CsA and cyclotides, one would expect that this effect can bereversed by exogenous addition of IL-2 to the treated cells. Therefore,PBMCs were treated with cyclotides and CsA and the cells were activatedwith PHA-L. In parallel, the cells were grown with addition of exogenousIL-2 (FIG. 13). Pretreatment of PBMCs with CsA and cyclotide T20K leadsto an anti-proliferative effect (13%±17.6 and 29%±24, respectively) ascompared to the control cells (FIG. 13A und B), whereas treatment withthe cyclotides V10A, V10K and T8K has no effect on the proliferation. Byadding exogenous IL-2 it was possible to reverse the anti-proliferativeeffect of CsA in part (54%±19.3) and of T20K almost completely (91%±1.4)(FIG. 13C und D). Addition of IL-2 to the V10A-, V10K- or T8K-treatedPBMC, did not change the effect on proliferation (FIG. 13).

EXAMPLE 11: INFLUENCE OF CYCLOTIDES ON THE IFN-GAMMA OR TNF-ALPHAPRODUCTION

From the results so far it is evident that treatment of activated PBMCswith CsA or cyclotide T20K influences the expression of the IL-2 surfacereceptor CD25 (FIG. 11) as well as the IL-2 secretion (FIG. 12).Furthermore, the anti-proliferative effect of T20K on PBMCs can beantagonized by addition of exogenous IL-2 (FIG. 13). Therefore it is ofinterest to determine whether cyclotides only have anti-proliferativeeffects or also affect the effector function of T-lymphocytes, whichwould directly relate to changes in the IFN-gamma and TNF-alphaproduction. Therefore, the production of both cytokines ofcyclotide-treated, activated PBMCs at an early time point after PBMCactivation was tested. PBMCs were pre-treated with either CsA orcyclotides followed by activation with PHA-L. After 24 h, the cells werere-stimulated for 6 h with PMA and ionomycin and afterwards theconcentrations of IFN-gamma (FIG. 14) and TNF-alpha (FIG. 15) in thecell supernatant was measured using an ELISA-based FACS method. TheIFN-gamma concentration of the CsA-treated cells was reduced to 14%±3.4as compared to the control and also the treatment with cyclotide T20Kyielded in an IFN-gamma reduction (21%±13.2). In summary, the IFN-gammaproduction after 24 h was significantly reduced by CsA (p≤0.01) and T20K(p≤0.001) (FIG. 14) but not by V10K (data not shown).

CsA (23%±1.8) and T20K (23%±10.6) also led to a significant (p.5_0.001)reduced TNF-alpha expression as compared to the control (FIG. 15). Totest whether the effector function of T-cells remains compromised aftertreatment with T20K we measured IFN-gamma and TNF-alpha release at alater time-point, i.e. 36 h past stimulation. The CsA-treated cellsexperienced a significant (p≤0.01) reduction in IFN-gamma production of23%±2 as compared to the control (FIG. 14) whereas all cyclotides (T20K,V10A, V10K, T8K) did not induce significant changes in the level ofIFN-gamma (FIG. 14). TNF-alpha production was significantly (p≤0.001)reduced after treatment with CsA (20%±14.4) whereas allcyclotide-treated cells did not result in any changes in the TNF-alphalevel (FIG. 15). Therefore it is obvious that treatment with cyclotideT20K leads to an initial reduction of the effector function, asindicated by the reduced IFN-gamma and TNF-alpha production, but thelevel of both cytokines stabilizes over time. This further indicatesthat T20K and CsA have different mechanism of action.

EXAMPLE 12: INFLUENCE OF CYCLOTIDES ON THE DEGRANULATION ACTIVITY OFACTIVATED PBMCS

After determining the influence of cyclotide treatment on the effectorfunction of PBMCs on the basis of measuring IFN-gamma and TNF-alphacytokine levels, it is of interest to determine an effect of cyclotideson the degranulation activity. Activation of cytotoxic CD8⁺-lymphocyteslead to a release of cytolytic granules, which contain/expresslysosomal-associated membrane protein 1 (CD107; LAMP-1). Duringdegranulation, the granule vesicle membranes fuse with the membranes ofactivated CD8⁺-lymphocytes and therefore LAMP-1 can be used as a markerprotein for the cytotoxic activity of T-lymphocytes, which can bemeasured with FACS. After 36 h, 42%±21.4 of the CsA- and 49%±16.8 of theT20K-treated cells contain the degranulation marker LAMP-1 as comparedto the control (FIG. 16). This can be interpreted in the way that CsA-and T20K-treated cells have reduced cytotoxicity. Cyclotides V10K andT8K had no influence on the degranulation activity of activated PBMCs(data not shown).

EXAMPLE 13: CA²⁺ RELEASE OF JURKAT CELLS

Jurkat cells T-cells were treated as described for FIG. 17, supra. ForJurkat cells CsA (5 mg/mL), T20K (4 μM) and V10K (4 μM) stimulation didnot induce a change in Ca²⁺ signaling in Jurkat cells. Since neither CsAnor cyclotides lead to any changes in Ca²⁺ signalling it is evident thateither compound will act downstream of Ca²⁺ release and hence thisindicates a similar immunosuppressive mechanism of cyclotides incomparison to CsA in these cells. In contrast human primary T-cellsdemonstrate an increasing Ca²⁺ release after incubation with thecyclotide T20K and hence the mechanism of action may be cell typedependent.

EXAMPLE 14: EFFECT OF CYCLOTIDES ON C57BL/6J MICE

Materials

Seven weeks old female C57BL/6J mice were purchased from the Departmentfor Lab-zoology and -genetics (Himberg, Austria). All experiments wereapproved according to the European Community rules of animal care withthe permission of the Austrian Ministry of Science. T20K and V10K wereprovided by D. J. Craik, from the University of Queensland, Institutefor Molecular Bioscience (Brisbane, Australia).5-carboxyfluoresceine-N-hydroxysuccinimid was purchased fromSigma-Aldrich (Vienna, Austria).

Immunization

Mice (n=10/group) were treated on day (−7), 0, 7 with 200 μg/100μL/mouse T20K solubilized in sterile PBS intraperitoneally (i.p.), asindicated in the figure. On day 0 they were immunized subcutaneouslywith myelin oligodendrocyte glycoprotein (MOG₃₅₋₅₅, 1 mg/mL) andcomplete Freud's adjuvant (CFA, 10 mg/mL) mixed at equal parts.Therefore 70 μL were injected into the left and right flank.Additionally mice received 100 μL pertussis toxin (2 μg/mL) i.p. on day0 and again on day 2. Beginning at day 10 mice were scored every secondday. Weight was also measured at day (−7), 0, 7 and on the same dayduring scoring. Mice were sacrificed on day 24 after reaching highscores.

Spleenocyte Isolation and Stimulation

Spleens of sacrificed mice were taken and transferred into a 6 cm PetriDish with 5 mL sterile PBS. To receive a spleenocyte suspension, spleenswere meshed and filtered through 70 μm nylon sieve. Cells werecentrifuged at 1200 rpm for 5 minutes and resuspended in RPMI 1640 mediasupplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine,penicillin (100 U/mL), and streptomycin (100 μg/mL). Spleenocytes werecultivated at a concentration of 3×10⁶/mL in a 48-well flat-bottom plate(500 μL/well). Cells were stimulated with 30 μg/mL MOG₃₅₋₅₅ or leftuntreated and incubated at 37° C. for three days. Supernatants and cellswere taken stored at −20° C. until further experiments. Cells isolatedfrom the naïve mouse group were additionally cultivated in a 96-wellflat-bottom plate (100 μL/well) and stimulated with T20K (4 μM),T20K+MOG, T20K (12 h), V10K (4 μM, 12 h), CsA (5 μg/mL; 12 h), MOG (12h) or left untreated. After 12 hours MOG or T20K was added toappropriate wells. Supernatants were stored after 24 hours and after 48hours at −20° C.

Enzyme Linked Immunosorbent Assay

Supernatants of stimulated spleenocytes were analyzed for their IL2,IL17, INFγ, IL4 and IL22 cytokine release using anti-mouse antibodiesfor ELISA from eBioscience according to the manufacturer's instructions.The color reaction was evaluated at an optical density of 450 nm by themicroplate reader Synergy H4 (BioTek).

SDS-PAGE and Western Blotting for NFAT1c

Human T-cells provided by CCRI from A. Dohnal, PhD were stimulatedaccording to the protocol for IL2 release described above. Stimulatedcells were resuspended in TBS and mixed at equal parts with samplebuffer and heated for five minutes at 95° C. A sodium dodecyl sulfatepolyacrylamide gel was prepared to separate the proteins achieved fromthe lysed T-cells. After electrophoresis proteins were transferred fromthe gel to a membrane. After blocking the membrane with BSA 3% in TBSTover night at 4° C., the first antibody mouse anti-NFATc1 was incubatedfor 2 hours at room temperature. After five times washing with TBST 0.1%Tween, the membrane was incubated with the second antibody anit-mouseIgG HRP for one hour at room temperature. The membrane was dried andtreated with West Pico or West Femto Super Signal Solution according tothe manufactures protocol to evaluate the chemo-luminescence signal.

EXAMPLE 15: CELL PERMEABILITY OF T20K CYCLOTIDES (CHEMICAL LABELLING ANDMICROSCOPY)

1.5 mg of T20K was dissolved in 1.5 ml of 100 mM sodium carbonate bufferof pH 8.8. 5-carboxyfluoresceine-N-hydroxysuccinimid ester (5-CFSE) wasadded in 10 fold excess as solid compound (2.5 mg) The reaction wasallowed to proceed for 120 min at room temperature. Afterwards thereaction mixture was heated to 50° C. for 30 min to complete thehydrolysis of the N-hydroxysuccinimid ester (NHS). Purification wasperformed using semi-preparative chromatography, applying a Kromasil RPcolumn 250×10 ID, 5 μm 100 Å. Eluent A was ddH₂O/TFA 99.9/0.1% (v/v),eluent B was AcN/H₂O/TFA 90/10/0.08% (v/v/v). The linear gradient from5% eluent B to 80% eluent B in 50 min was used. Maldi-TOF-MS analysis ofthe collected fractions yielded a mass of 3276.3 Da in one of thefractions. The mass peak of 3276.3 Da were identified as themono-derivatized species of T20K with 5-carboxyfluoresceine with a massshift of 357 Da. Human T-cells and Jurkat cells were incubated with a 4μM solution of the T20K derivative in RPMI 1640 media supplemented withthe additives described above for 20 min. The fluorescence microscopewas from Zeiss LSM 510 confocal microscope. The excitation wavelengthwas 488 nm and emission wavelength 520 nm.

EXAMPLE 16

The present invention refers to the following supplemental tables:

TABLE 1 Cyclotides from O. affinis extract identified by nano LC-MS andMS/MS Theoretical MW (avg.) MW (mono.) MW Δ MW Cyclotide¹ Da² Da² Score³Evidence⁴ Da⁵ Da⁶ kalata B1 2892.85 2890.39 1 ICP 2892.33 0.52 kalata B22956.14 2953.74 1 ICS 2955.38 0.76 kalata B3 3083.31 3080.64 1 ICS3082.48 0.83 kalata B4 2893.24 2890.56 1 IS 2893.31 0.07 kalata B5 — — —P 3061.59 — kalata B6 3029.96 3027.66 0.9999 IS 3029.42 0.54 kalata B73072.26 3069.74 0.9998 IS 3071.59 0.67 kalata B8 3284.34 3281.75 1 ICS3283.79 0.55 kalata B9 — — — P 3272.72 — kalata B9 lin — — — P 3290.74 —kalata B10 3030.21 3027.53 1 ICS 3030.41 0.20 kalata B10 lin 3048.543046.50 1 ICS 3048.43 0.11 kalata B11 2884.48 2881.44 0.9999 I 2884.260.22 kalata B12 — — — P 2880.27 — kalata B13 3036.06 3033.58 1 IC3036.46 0.40 kalata B14 3023.74 3021.17 0.9987 I 3022.43 1.31 kalata B152977.00 2974.56 1 ICS 2976.40 0.60 kalata B18 3147.33 3145.02 0.9977 I3145.67 1.66 kalata S 2878.81 2875.93 0.9993 I 2878.30 0.51 Oak6cyclotide 1 3035.87 3033.49 1 IC 3035.47 0.40 [G-A] kalata B1⁷ 2906.472904.75 0.9995 I 2906.35 0.12 kalata b1-1 2724.12 2722.28 1 IC 2724.180.06 [L2A] kalata B1 2851.88 2849.54 1 IC 2850.25 1.63Ac-[desGly]-KB1-Am 2854.31 2851.68 0.9996 I 2853.30 1.01 acyclic kalataB1 2911.32 2908.36 1 IC 2910.35 0.97 Oak6 cyclotide 2 3093.29 3090.61 1IC 3092.56 0.73 ¹Identification by LC-MS reconstruct of at least 3representative LC-MS experiments (±1 Da, 20-70 min, EMS 1000 2 scans) oridentification by digest (trypsin or endo-GluC), nano LC-MS/MS anddatabase search (ERA); ²Observed molecular weight; ³Score indicating thequality of LC-MS reconstructed peptide MW (≤1.0); ⁴Evidence foridentified cyclotides, I = isotope pattern, C = charge pattern, S = fullsequence, P = partial sequence or sequence tag; ⁵Data taken from CyBase(Wang, 2008, Nucleic Acids Res, 36, D206-210); ⁶Δ MW determined toaverage molecular weight; ⁷amino acid position (G-A replacement) notspecified

TABLE 3 Comparison of kalata B1 (and other cyclotides) inhibitoryeffects IC50 values in various cellular test systems. Assay system CellsIC₅₀ (μM) Reference kalata B1 Anti-proliferative activity humanperipheral blood 3.9 ± 0.5 Gründemann et al., mononuclear cells 2012Nematocidal activity H. contortus nematodes 2.7 Huang et al., 2010 T.colibriformus nematodes 4.5 Huang et al., 2010 Cytotoxicity humanT-lymphoblast cells 3.5 Daly et al., 2004 other cyclotides* Cytotoxicityhuman lymphoma cell line 0.6-6 Svangard et al., 2004 (U-937) 0.3-7Lindholm et al., 2002 Cytotoxicity human myeloma cell line   1-4Svangard et al., 2004 (RPMI-8226/s) 0.1-6 Lindholm et al., 2002*Activity was reported of various cyclotides (varv A, varv E, varv F,vitri A, cycloviolacin O2) from Viola arvensis, V. odorata and V.tricolor

TABLE 4 LC-MS reconstruct of O. affinis cyclotides. Raw (labelled) dataof LC-MS reconstruct of O. affinis extracts as analysed by nano LC-MS.Mass Theoretical Δ Da Da Mass Mass No. cyclotide (avg.) (mono.) ScoreEvidence Da (avg.) Da  1 new 2706.63 2704.37 0.9997 I  2 new 2723.222721.27 1 I  3 kalata b1-1 2724.12 2722.28 1 IC 2724.18 0.0559  4 new2821.78 2819.36 1 IC  5 new 2822.30 2820.55 0.9995 I  6 new 2833.302831.41 1 I  7 [L2A] kalata B1 2851.88 2849.54 1 IC 2850.25 1.6322Ac-[desGly]-KB1-  8 Am 2854.31 2851.68 0.9996 I 2853.3 1.0072  9 new2873.73 2871.13 0.9996 I 10 kalata S 2878.81 2875.93 0.9993 I 2878.300.5141 11 new 2879.82 2877.46 1 IC 12 kalata B12** 2882.30 2880.830.9969 I 2880.27 2.0256 13 kalata B11 2884.48 2881.44 0.9999 I 2884.260.2236 14 new 2891.45 2888.50 1 IC 15 kalata B1 2892.85 2890.39 1 IC2892.33 0.5228 16 kalata B4 2893.24 2890.56 1 I 2893.31 0.0718 17 new*2896.70 2894.45 0.9999 I 18 new 2897.11 2894.57 1 IC 19 [G-A] kalata B12906.47 2904.75 0.9995 I 2906.35 0.1191 20 new 2909.53 2906.90 1 IC 21acyclic kalata B1 2911.32 2908.36 1 IC 2910.35 0.9675 22 new* 2912.902911.43 0.9999 I 23 new* 2922.95 2920.48 1 IC 24 new* 2925.32 2922.40 1IC 25 new* 2926.80 2923.70 1 IC 26 new 2927.30 2924.66 1 IC 27 new2937.91 2935.50 0.9998 I 28 new 2942.99 2940.48 1 IC 29 kalata B22956.14 2953.74 1 IC 2955.38 0.7637 30 new* 2959.95 2957.56 1 IC 31 new2960.36 2958.24 0.9996 I 32 new 2969.25 2968.13 0.9997 I 33 new* 2971.442969.50 1 IC 34 new 2973.80 2970.55 1 IC 35 new 2974.14 2971.51 1 IC 36new* 2975.38 2973.49 1 IC 37 kalata B15 2977.00 2974.56 1 IC 2976.400.602  38 new* 2986.57 2983.80 1 I 39 new 2988.28 2985.60 1 IC 40 new2990.37 2987.51 1 IC 41 new 2994.11 2991.86 1 IC 42 new* 3006.25 3003.501 IC 43 new* 3010.97 3008.88 1 IC 44 kalata B14 3023.74 3021.17 0.9987 I3022.43 1.3147 45 new* 3028.61 3025.92 0.9998 I 46 kalata B6 3029.963027.66 0.9999 I 3029.42 0.5381 47 kalata B10 3030.21 3027.53 1 IC3030.41 0.2028 48 Oak6 cyclotide 1 3035.87 3033.49 1 IC 3035.47 0.398 49 kalata B13 3036.06 3033.58 1 IC 3036.46 0.4018 50 new 3039.91 3037.451 IC 51 new 3040.05 3036.62 1 IC 52 new* 3045.78 3043.50 1 IC 53 new*3046.32 3044.95 1 IC 54 new 3047.97 3046.60 0.9999 I 55 kalata B10 lin3048.54 3046.50 1 IC 3048.43 0.1091 56 new* 3051.82 3048.48 1 IC 57 new*3052.72 3049.57 1 IC 58 new* 3065.79 3063.36 0.9997 I 59 kalata B73072.26 3069.74 0.9998 I 3071.59 0.67  60 new* 3073.89 3072.70 0.9999 I61 kalata B3 3083.31 3080.64 1 IC 3082.48 0.8309 62 new* 3087.22 3084.611 IC 63 new 3089.27 3086.96 0.9997 I 64 new 3091.00 3089.03 0.9997 I 65Oak6 cyclotide 2 3093.29 3090.61 1 IC 3092.56 0.7328 66 new* 3097.633094.57 1 IC 67 new* 3099.85 3096.60 1 IC 68 kalata B18** 3147.333145.02 0.9977 I 3145.67 1.6615 69 new* 3266.81 3264.99 0.9997 I 70kalata B8 3284.34 3281.75 1 IC 3283.79 0.5453 71 new* 3300.96 1 C 72 new3446.88 3444.98 0.9998 I Total: 72 New: 25 New*: 24 LC-MS reconstruct,EMS 1000 Da/sec Reconstruct 2700-3500 Da, signal-to-noise: 4, 25, 50;combined datasets from at least 3 independent LC-MS runs CyBasecomparison: MW +/− 1 Da * = other cylotide detected (not Oaffinis) ** =+/− Da

TABLE 5 O. affinis database search results following digests andLC-MS/MS analysis. Protein Pilot ™ database search results of thecyclotide LC-MS/MS analysis. % % N Unused Total % Cov Cov(50) Cov(95)Accession Name Peptides(95%) trypsin digest 1 6.43 6.43 100.00 85.4882.26 cb|P85175 kalata B8/1-31|cybaseid = 168 organism = Oldenlandiaaffinis 4 2 6.00 6.00 100.00 51.72 51.72 cb|P58457 kalataB7/1-29|cybaseid = 26 organism = Oldenlandia affinis 6 3 5.91 5.91100.00 98.28 96.55 cb|P58454 kalata 62/1-29|cybaseid = 4 organism =Oldenlandia affinis 4 4 2.75 2.75 100.00 98.28 72.41 cb|P83938 kalataB411-29|cybaseid = 30 organism = Oldenlandia affinis 2 5 2.63 2.63 93.3388.33 88.33 cb|P58456 kalata B5/1-30|cybaseid = 59 organism =Oldenlandia affinis 5 6 2.00 3.75 100.00 98.28 72.41 cb|P85133 kalataB15/1-29|cybaseid = 253 organism = Oldenlandia affinis 3 7 2.00 2.00100.00 50.00 50.00 cb|P85128 kalata B10/1-30|cybaseid = 246 organism =Oldenlandia affinis 1 7 0.00 2.00 100.00 50.00 50.00 cb|P247 kalata B10linear/1-30|cybaseid = 247 organism = 1 Oldenlandia affinis 8 2.00 2.0085.48 85.48 43.55 cb|P85127 kalata B9/1-31|cybaseid = 244 organism =Oldenlandia affinis 1 8 0.00 2.00 85.48 85.48 43.55 cb|P245 kalata 69linear/1-31|cybaseid = 245 organism = 1 Oldenlandia affinis 9 2.00 2.0098.21 50.00 50.00 cb|P85130 kalata B12/1-28|cybaseid = 250 organism =Oldenlandia affinis 2 10 1.06 2.00 100.00 50.00 50.00 cb|P58455-b3kalata B6/1-30|cybaseid = 24 organism = Oldenlandia affinis 1 10 0.002.00 100.00 50.00 50.00 cb|P247 kalata B10 linear/1-30cybaseid = 247organism = 1 Oldenlandia affinis 11 0.63 2.01 98.28 98.28 72.41cb|P56254 kalata B1/1-29|cybaseid = 1 organism = Viola odorata; 4Oldenlandia affinis; Viola baoshanensis; Viola yedoensis 12 0.20 0.20100.00 61.67 0.00 cb|P58455-b6 kalata B3/1-30|cybaseid = 25 organism =Oldenlandia affinis 0 Endo GluC digest 1 0.88 0.88 100 87.92999983 0cb|P58454 kalata B2/1-29|cybaseid = 4 organism = Oldenlandia affinis 0 20.52 0.52 100 50 50 cb|P58457 kalata B7/1-29|cybaseid = 26 organism =Oldenlandia affinis 1 3 0.21 0.21 100 25 0 cb|P58455-b6 kalataB3/1-30|cybaseid = 25 organism = Oldenlandia affinis 0

TABLE 6 Cyclotide quantification data. Data of five independentexperiments of cyclotide quantification. MW MW LC-MS MW Identifiedcalculated Δ MW reconstruct Δ MW theoretical RT Area Height Rel.Areacyclotide (Da) SEM (Da) (Da) SEM (Da) (Da) (min) SEM (mAU*min) SEM (mAU)SEM % SEM kalata B8 3283.91 0.15 0.12 328424 0.19 0.45 3283.79 29.950.01 20.55 0.57 28.01 0.72 5.6 0.2 kalata B7 3071.83 0.19 0.24 3072.270.17 0.66 3071.59 37.54 0.04 7.89 0.22 41.43 0.21 2.2 0.1 kalata B12892.27 0.27 0.06 2892.98 0.21 0.65 2892.33 45.48 0.02 50.81 0.84 96.960.61 13.9 0.2 kalata B6 3029.60 0.28 0.18 3029.84 0.12 0.42 3029.4246.50 0.03 29.63 0.46 46.88 0.38 0.1 0.1 kalata B13 3035.56 0.20 0.90303589 0.12 0.57 3036.46 50.32 0.03 14.88 1.16 28.75 0.38 4.1 0.3 kalataB2 2955.76 0.12 0.38 2955.90 0.09 0.52 2955.38 51.82 0 03 72.86 3.17103.65 0.86 20.0 0.6 kalata B3 3082.72 0.21 0.24 3083.04 0.08 0.553082.48 52.59 0.07 11.00 1.40 23.02 1.27 3.0 0.4 *n = 5, HPLCquantification (area under curve) of five independent experiments **MWaverage mass ***MW calculated from +2 or +3 ion

The present invention refers to the following nucleotide and amino acidsequences:

SEQ ID No. 1: Amino acid sequence of Kalata B1:GLPVCGETCVGGTCNTPGCTCSWPVCTRN SEQ ID No. 2: Amino acid sequence ofKalata B2: GLPVCGETCFGGTCNTPGCSCTWPICTRD SEQ ID No. 3: Amino acidsequence of D-Kalata B2: all-D GLPVCGETCFGGTCNTPGCSCTWPICTRD SEQ ID No.4: Amino acid sequence of Kalata G18K: GLPVCGETCVGGTCNTPKCTCSWPVCTRN SEQID No. 5: Amino acid sequence of Kalata N29K:GLPVCGETCVGGTCNTPGCTCSWPVCTRK SEQ ID No. 6: Amino acid sequence ofKalata T20K, G1K: KLPVCGETCVGGTCNTPGCKCSWPVCTRN SEQ ID No. 7: Amino acidsequence of Kalata T20K: GLPVCGETCVGGTCNTPGCKCSWPVCTRN SEQ ID No. 8:Amino acid sequence of Kalata T8K: GLPVCGEKCVGGTCNTPGCTCSWPVCTRN SEQ IDNo. 9: Amino acid sequence of Kalata V10A: GLPVCGETCAGGTCNTPGCTCSWPVCTRNSEQ ID No. 10: Amino acid sequence of Kalata V10K:GLPVCGETCKGGTCNTPGCTCSWPVCTRN SEQ ID No. 11: Nucleotide sequenceencoding Kalata B1: GGACTTCCAGTATGCGGTGAGACTTGTGTTGGGGGAACTTGCAACACTCCAGGCTGCACTTGCTCCTGGCCTGTTTGCACACGCAAT SEQ ID No. 12: Nucleotide sequenceencoding Kalata B2: GGTCTTCCAGTATGCGGCGAGACTTGCTTTGGGGGAACTTGCAACACTCCAGGCTGCTCTTGCACCTGGCCTATCTGCACACGCGAT SEQ ID No. 13: Amino acid sequenceof the Kalata B1 precursor protein. The mature Kalata B1 domain isunderlined. P56254, Kalata-B1, Oldenlandia affinisMAKFTVCLLLCLLLAAFVGAFGSELSDSHKTTLVNEIAEKMLQRKILDGVEATLVTDVAEKMFLRKMKAEAKT SETADQVFLKQLQLK GLPVCGETCVG GTCNTPGCTCSWPVCTRNGLPSLAA SEQ ID No. 14: Amino acid sequence of the Kalata B2 precursorprotein. The three mature Kalata B2 domains are underlined. P58454,Kalata-B2, Oldenlandia affinisMAKFTNCLVLSLLLAAFVGAFGAEFSEADKATLVNDIAENIQKEILGEVK TSETVLTMELKEMQLKGLPVCGETCFGGTCNTPGCSCTWPICTRD SLPMR AGGKTSETTLHMFLKEMQLKGLPVCGETCFGGTCNTPGCSCTWPICTRD S LPMSAGGKTSETTLHMELKEMQLKGLPVCGETCFGGTCNTPGCSCTWPIC TRD SLPLVAA SEQ ID No. 15: Nucleotidesequence encoding the Kalata B1 precursor protein. The nucleotidesequence corresponding to the mature Kalata B1 domain is underlined.>gi|15667740|gb|AF393825.1|Oldenlandia affinis kalata B1 precursor,mRNA, complete cds GGCACCAGCACTTTCTTAAAATTTACTGCTTTTTCTTATTTCTTGTTCTGTGCTTGCTTCTTCCATGGCTAAGTTCACCGTCTGTCTCCTCCTGTGCTTGCTTCTTGCAGCATTTGTTGGGCCGTTTGGATCTGAGCTTTCTGACTCCCACAAGACGACCTTGGTCAATGAAATCGCTGAGAAGATGCTACAAAGAAAGATATTGGATGGAGTGGAAGCTACTTTGGTCACTGATGTCCCCGAGAAGATGTTCCTAAGAAAGATGAAGGCTGAAGCGAAAACTTCTGAAACCGCCGATCAGGTGTTCCTGAAACAGTTGCAGCTCAAAGGACTTCCACTATGCGGTGAGACTTGTGTTGGCGCAACTTGCAACACTCCAGGCTCCACTTGCTCCTGGCCTGTTTGCACACGCAATGGCCTTCCTAGTTTGGCCGCATAATTTGCTTGATCAAACTGCAAAAATGAATGAGAAGGCCGACACCAATAAAGCTATCAATGTAGTTGGTCCCTGTACTTAATTTGGTTGGCTCCAAACCATGTGTGCTGCTCTTGTTTTTGTTTTTTCTTTTTTCTTCTCTCTTTCGGGCACTCTTCAGGACATGAAGTGATGATCAGTACTCTTTGCTATCATGTTTTCTGTGCACACCTTCTATTGTAGGTGTTGTTGTGATGTTGATGCCCAATTGGATAACTGTTGTCG TTGTTAAAAAAAAAAAAAASEQ ID No. 16: Nucleotide sequence of encoding the Kalata B2 precursorprotein. The nucleotide sequences corresponding to the three matureKalata B2 domais are underlined. >gi|15667746|gb|AF393828.1|Oldenlandiaaffinis kalata 82 precursor, mRNA, complete cdsGGCACCAGATACAACCCCTTTCTTATAATTTATTGCTTTTCTTATTCCTTGAAAAAGGAGAAATAATATTGGATCTTCCATGGCTAAGTTCACCAACTGTCTCGTCCTGAGCTTGCTTCTAGCAGCATTTGTTGGGGCTTTCGGAGCTGAGTTTTCTGAAGCCGACAAGGCCACCTTGGTCAATGATATCGCTGAGAATATCCAAAAAGAGATACTGGGCGAAGTGAAGACTTCTGAAACCGTCCTTACGATGTTCCTGAAAGAGATGCAGCTCAAAGGTCTTCCAGTATGCGGCGAGACTTGCTTTGGGGGAACTTGCAACACTCCAGGCTGCTCTTGCACCTGGCCTATCTGCACACGCGATAGCCTTCCTATGAGGGCTGGAGGAAAAACATCTGAAACCACCCTTCATATGTTCCTGAAAGAGATGCAGCTCAAGGGTCTTCCAGTTTGCCGCGAGACTTGCTTTGGGGGAACTTGCAACACTCCAGGCTGCTCGTGCACCTGGCCTATCTGCACACGCGATAGCCTTCCTATGAGTGCTGGAGGAAAAACATCTGAAACCACCCTTCATATGTTCCTGAAAGAGATGCAGCTCAAGGGTCTTCCAGTTTGCGGCGAGACTTGCTTTGGGGGAACTTGCAACACTCCAGGCTGCTCGTGCACCTGGCCTATATGCACACGTGATAGCCTTCCTCTTGTGGCTGCATAATTTGCTTCATCAAACTGCAAAATGAATAAGAAGGGACACTAAATTAGCTATGAATTTTGTTGGCCCTTGTGTCTGGTAATTTGGTTCCCGCCAAATTAACCATATGTATGCATTGCTCCTTTTTTCTTTCTTTTTTTTCCCCCTCATTTGGGCACTCTTCATTACATGAAGAGATCATGACGCTTTGTTACTCTGAGCACCCCCTGTTGGTGTTGTTCACATGTTGATGCCCATGTTGGAATAAACTCTTGTTTTTGTTACCAAAAAAAAAAAAAAAAA SEQ ID No. 17: Consensus aminoacid sequence of active Cyclotides (Xxx₁ is any amino acid, non-naturalamino acid or peptidomimetic; Xxx₂ is any amino acid, non- natural aminoacid or peptidomimetic but not Lys; and Xxx₃ is any amino acid,non-natural amino acid or peptidomimetic but not Ala or Lys):Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Xxx₁-Cys-Xxx₁-Trp-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁

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The invention claimed is:
 1. Method of screening for and/or selecting animmunosuppressive cyclotide, the method comprising: (i) contactingactivated immune cells with a cyclotide or a plant extract containing acyclotide and determining the proliferative activity of said activatedimmune cells, wherein a reduced proliferative activity as compared to acontrol is indicative for the immunosuppressive activity of thecyclotide or plant extract containing a cyclotide; or (ii) administeringto an animal model a pharmaceutically effective amount of a cyclotide ora plant extract containing a cyclotide and determining one or moreparameters of the immune system, wherein the reduction of the one ormore parameters of the immune system as compared to a control isindicative for the immunosuppressive activity of the cyclotide or plantextract containing a cyclotide.
 2. The method of claim 1, comprisingintroducing a mutation into the cyclotide prior to: contacting theactivated immune cells with the cyclotide or plant extract; oradministering to the animal model a pharmaceutically effective amount ofthe cyclotide or plant extract.
 3. The method of claim 2, wherein themutated cyclotide demonstrates: a reduced proliferative activity inactivated immune cells as compared to the non-mutated cyclotide; or areduction of one or more parameters of the immune system in the animalmodel as compared to the non-mutated cyclotide.
 4. The method of claim1, further comprising isolating and/or identifying the immunosuppressivecyclotide from the plant extract.
 5. The method of claim 1, wherein thecyclotide is or comprises a non-grafted cyclotide.
 6. The method ofclaim 1 (i), wherein the control comprises: determining theproliferative activity of activated immune cells that have not beencontacted with the cyclotide or the plant extract containing acyclotide; or determining the proliferative activity of activated immunecells that have been contacted with a known cyclotide that does not haveimmunosuppressive activity.
 7. The method of claim 1 (ii), wherein thecontrol comprises: determining the one or more parameters of the immunesystem in the animal model that have not been administered the cyclotideor the plant extract containing a cyclotide; or determining the one ormore parameters of the immune system in the animal model that have beenadministered a known cyclotide that does not have immunosuppressiveactivity.
 8. The method of claim 1 (i), wherein activated immune cellscomprise activated human peripheral blood mononuclear cells (PBMCs) orhuman T-lymphocyte.
 9. The method of claim 8, wherein the activatedhuman PBMCs comprises CD107a⁺ CD8⁺ PBMCs.
 10. The method of claim 1(ii), wherein the parameter of the immune system is selected from thegroup consisting of secretion/production of IL-2, secretion/productionof IFN-gamma, secretion/production of TNF-alpha,degranulation/cytotoxicity of CD107a+ CD8+ PBMCs, and expression of IL-2surface receptor CD25.
 11. The method of claim 1 (ii), wherein theanimal model comprises an animal model for an autoimmune disease. 12.The method of claim 11, wherein the animal model for an autoimmunedisease comprises the Experimental Autoimmune Encephalitis (EAE) mousemodel for Multiple Sclerosis (MS).
 13. The method of claim 1, whereinthe cyclotide comprises a head-to-tail cyclized peptide comprising sixconserved cysteine residues capable of forming three disulphide bondsarranged in a cyclic cystine knot (CCK) motif.
 14. The method of claim1, wherein said cyclotide comprises a cyclic backbone having thestructure of formula I, wherein formula I comprises the amino acidsequence:Cyclo(C[X₁ . . . Xa] C[XI1 . . . XIb] C[XII1 . . . XIIc] C[XIII1 . . .XIIId] C[XIV1 . . . XIVe] C[XV1 . . . XVf]); wherein C is cysteine;wherein each of [X1 . . . Xa], [XI1 . . . XIb], [XII1 . . . XIIc],[XIII1 . . . XIIId], [XIV1 . . . XIVe], and [XV1 . . . XVf] representsone or more amino acid residues, wherein each one or more amino acidresidues within or between the sequence residues may be the same ordifferent; and wherein a, b, c, d, e, and f represent the number ofamino acid residues in each respective sequence and each of a to f maybe the same or different and range from 1 to about
 20. 15. The method ofclaim 14, wherein a is 3 to 6, b is 4 to 8, c is 3 to 10, d is 1, e is 4to 8, and/or f is 5 to
 13. 16. The method of claim 1, wherein thecyclotide is or comprises a mutant or variant of kalta B.
 17. The methodof claim 1, wherein said cyclotide comprises an amino acid sequence offormula II; wherein formula II comprises: (SEQ ID NO. 17)Xxx₁-Leu-Pro-Val-Cys-Gly-Glu-Xxx₂-Cys-Xxx₃-Gly-Gly-Thr-Cys-Asn-Thr-Pro-Xxx₁-Cys-Xxx₁-Cys-Xxx₁-Trp-Pro-Xxx₁-Cys-Thr-Arg-Xxx₁,;

wherein Xxx₁ comprises any amino acid, non-natural amino acid orpeptidomimetic; wherein Xxx₂ comprises any amino acid, non-natural aminoacid or peptidomimetic but not Lys; and wherein Xxx₃ comprises any aminoacid, non-natural amino acid or peptidomimetic but not Ala or Lys. 18.The method of claim 1, wherein said cyclotide comprises an amino acidsequence that is at least 90% identical to any amino acid sequence ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, and
 7. 19. The method of claim 1, whereinsaid cyclotide reduces secretion/production of IL-2, reducessecretion/production of IFN-gamma, reduces secretion/production ofTNF-alpha, suppresses/reduces degranulation/cytotoxicity of CD107a+CD8+PBMCs, and/or suppresses/reduces expression of IL-2 surface receptorCD25.
 20. The method of claim 1, wherein the anti-proliferative effector suppression/reduction is mediated in an IL-2-, IFN-gamma- and/orTNF-alpha-depending manner and/or can be antagonized by IL-2.